Stem cells of the islets of langerhans and their use in treating diabetes mellitus

ABSTRACT

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin has been identified as a molecular marker for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

TECHNICAL FIELD OF THE INVENTION

[0001] The invention is related to the field of stem cells and theirdifferentiation. In particular, it is related to the field of beta cellsof the islets of Langerhans in the pancreas and nestin positive liverstem cells and their differentiation from stem cells or progenitorcells, and the use of pancreatic stem cells, progenitor cells, anddifferentiated beta cells or nestin positive liver stem cells orprogenitor cells in transplantation.

BACKGROUND OF THE INVENTION

[0002] The invention was made at least in part using U.S. governmentfunds, grants DK30457 and DK30834 awarded by the National Institutes ofHealth, and therefore the U.S. government may retain certain rights inthe invention.

[0003] The origin of pancreatic islet cells, both during embryonicdevelopment and in a mature mammal, has remained uncertain despiteintensive study. Certain ductal epithelial cells are capable of eitherdifferentiation or transdifferentiation to form beta cells and othercell types found in mature islets (Bouwens, 1998). Ductal cells fromisolated islets can proliferate in culture and, if transplanted into ananimal, can differentiate into functional beta cells (Cornelius et al.,1997).

[0004] It has been demonstrated that exendin-4, a long acting GLP-1agonist, stimulates both the differentiation of β-cells from ductalprogenitor cells (neogenesis) and proliferation of β-cells whenadministered to rats. In a partial pancreatectomy rat model of type 2diabetes, the daily administration of exendin-4 for 10 days postpancreatectomy attenuated the development of diabetes. It has also beendemonstrated that exendin-4 stimulates the regeneration of the pancreasand expansion of β-cell mass by neogenesis and proliferation of β-cells(Xu et al., 1999, Diabetes, 48:2270-2276).

[0005] Ramiya et al. have demonstrated that islets generated in vitrofrom pluripotent stem cells isolated from the pancreatic ducts of adultprediabetic non-obese diabetic (NOD) mice differentiate to form glucoseresponsive islets that can reverse insulin-dependent diabetes afterbeing implanted, with or without encapsulation, into diabetic NOD mice(Ramiya et al., 2000, Nature Med., 6:278-282).

[0006] The insulinotropic hormone glucagon-like peptide (GLP)-1 which isproduced by the intestine, enhances the pancreatic expression of thehomeodomain transcription factor IDX-1 that is critical for pancreasdevelopment and the transcriptional regulation of the insulin gene.Concomitantly, GLP-1 administered to diabetic mice stimulates insulinsecretion and effectively lowers their blood sugar levels. GLP-1 alsoenhances β-cell neogenesis and islet size (Stoffers et al., 2000,Diabetes, 49:741-748).

[0007] Ferber et al. have demonstrated that adenovirus-mediated in vivotransfer of the PDX-1 (also known as IDX-1) transgene to mouse liverresults in the transconversion of a hepatocyte subpopulation towards aβ-cell phenotype. It has been demonstrated that after intravenousinfusion of mice with the PDX-1 adenoviral vector, up to 60% ofhepatocytes synthesized PDX-1. The concentration of immunoreactiveinsulin was increased in the liver and serum of treated mice. Micetreated with PDX-1 survive streptozotocin-induced diabetes, and can evennormalize glycemia (Ferber et al., 2000, Nature Med., 6:568-572).

[0008] While ductal cell cultures obtained from isolated isletsapparently contain cells that can give rise to insulin-secreting cells,it has remained unclear whether those cells represent true stem cells ormerely ductal epithelial cells undergoing transdifferentiation. Even ifsuch preparations contain genuine stem cells, it is unknown whatfraction represent stem cells and what contaminating cell types may bepresent. There is a need in the art for the isolation of specific celltypes from pancreatic tissue, the cell types being characterized as stemcells using molecular markers and demonstrated to be pluripotent and toproliferate long-term.

[0009] Pluripotent stem cells that are capable of differentiating intoneuronal and glial tissues have been identified in brain. Neural stemcells specifically express nestin, an intermediate filament protein(Lendahl et al., 1990; Dahlstrand et al., 1992). Nestin is expressed inthe neural tube of the developing rat embryo at day E11, reaches maximumlevels of expression in the cerebral cortex at day E 16, and decreasesin the adult cortex, becoming restricted to a population of ependymalcells (Lendahl et al., 1990). Developing neural and pancreatic isletcells exhibit phenotypic similarities characterized by common cellularmarkers.

[0010] The invention relates to a population of pancreatic isletstem/progenitor cells (IPCs) that are similar to neural and hepatic stemcells and differentiate into islet o-cells (glucagon) and β-cells(insulin). The invention also relates to nestin-positive liver cells.IPCs according to the invention are immunologicallysilent/immunoprivileged and are recognized by a transplant recipient asself. The IPCs according to the invention can be used for engraftmentacross allogeneic and xenogeneic barriers.

[0011] There is a need in the art for a method of engrafting stem cellsacross allogeneic and xenogeneic barriers.

[0012] There is also a need in the art for a method of treating type Idiabetes mellitus wherein islets, nestin-positive pancreatic stem cellsor nestin-positive liver stem cells are transferred into a recipientacross allogeneic or xenogeneic barriers and graft rejection does notoccur.

[0013] There is also a need in the art for a method of transplantationinto a mammal wherein islets, nestin-positive pancreatic stem cells ornestin-positive liver stem cells are transferred into a recipient acrossallogeneic or xenogeneic barriers and graft rejection does not occur.

SUMMARY OF THE INVENTION

[0014] It is an object of the invention to provide mammalian pancreaticor liver stem cells for use in treating diabetes mellitus and otherdisorders. It is also an object of the invention to provide methods foridentifying, localizing, and isolating pancreatic stem cells. It is afurther object of the invention to provide methods for differentiatingpancreatic stem cells to obtain cells that produce insulin and otherhormones. It is also an object of the invention to provide methods fortransplantation into a mammal that utilize mammalian pancreatic or liverstem cells. These and other objects of the invention are provided by oneor more of the embodiments described below.

[0015] One embodiment of the invention provides a method of treating apatient with diabetes mellitus. A nestin-positive pancreatic stem cellis isolated from a pancreatic islet of a donor. The stem cell istransferred into the patient, where it differentiates into aninsulin-producing cell.

[0016] Another embodiment provides another method of treating a patientwith diabetes. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor and expanded ex vivo to produce a progenitorcell. The progenitor cell is transferred into the patient, where itdifferentiates into an insulin-producing beta cell. Another embodimentprovides still another method of treating a diabetes patient. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and expanded to produce a progenitor cell. The progenitorcell is differentiated in culture to form pseudo-islet like aggregatesthat are transferred into the patient.

[0017] Another embodiment provides another method of treating a patientwith diabetes mellitus. A nestin-positive pancreatic stem cell isisolated from a pancreatic islet of a donor and cultured ex vivo toproduce a progenitor cells. The progenitor cell is transferred into thepatient, where it differentiates into an insulin-producing beta cell.

[0018] In these embodiments, the patient can also serve as the donor ofthe pancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

[0019] In another preferred embodiment, prior to the step oftransferring, the stem cell is treated ex vivo with an agent selectedfrom the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF,GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1,betacellulin, activin A, TGF-β, and combinations thereof.

[0020] In another preferred embodiment, the step of transferring isperformed via endoscopic retrograde injection.

[0021] In another preferred embodiment, the method of treating a patientwith diabetes mellitus additionally comprises the step of treating thepatient with an immunosuppressive agent.

[0022] In another preferred embodiment, the immunosuppressive agent isselected from the group consisting of FK-506, cyclosporin, and GAD65antibodies.

[0023] Another embodiment provides a method of isolating a stem cellfrom a pancreatic islet of Langerhans. A pancreatic islet is removedfrom a donor, and cells are cultured from it. A nestin-positive stemcell clone is selected from the culture. Optionally, the islet is firstpurged of non-islet cells by culturing in a vessel coated withconcanavalin A, which binds the non-islet cells.

[0024] In a preferred embodiment, the method of isolating a stem cellfurther comprises the additional step of expanding the nestin-positiveclone by treatment with an agent selected from the group consisting ofEGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, anucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β,and combinations thereof.

[0025] A further embodiment provides a method of inducing thedifferentiation of a nestin-positive pancreatic stem cell into apancreatic progenitor cell. As used herein, “differentiation” refers tothe process by which a cell undergoes a change to a particular celltype, e.g. to a specialized cell type. The stem cell is treated with anagent selected from the group consisting of EGF, bFGF-2, high glucose,KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1,exendin-4, betacellulin, activin A, TGF-β, and combinations thereof. Thestem cell subsequently differentiates into a pancreatic progenitor cell.

[0026] In a preferred embodiment, the pancreatic progenitor subsequentlyforms pseudo-islet like aggregates.

[0027] Yet another embodiment provides an isolated, nestin-positivehuman pancreatic or liver stem cell. In versions of this embodiment, thestem cell differentiates into either a beta cell, an alpha cell, apseudo-islet like aggregate, or a hepatocyte. In versions of thisembodiment, the stem cell is immunoprivileged. In versions of thisembodiment, the stem cell does not express class I MHC antigens. Inversions of this embodiment, the stem cell does not express class II MHCantigens. In versions of this embodiment, the stem cell does not expressclass I or class II MHC antigens.

[0028] Still another embodiment provides a method of identifying apancreatic cell as a stem cell. A cell is contacted with a labelednestin-specific antibody. If the cell becomes labeled with the antibody,then the cell is identified as a stem cell. Optional additional stepsinclude contacting the cell with an antibody to cytokeratin 19 and anantibody to collagen IV; the cell is identified as a stem cell if itdoes not become labeled with either the cytokeratin 19 or the collagenIV antibody.

[0029] Another embodiment provides a method of inducing anestin-positive pancreatic stem cell to differentiate into hepatocytes.The nestin-positive pancreatic stem cell is treated with an effectiveamount of an agent that induces the stem cell to differentiate intohepatocytes or into progenitor cells that differentiate intohepatocytes. In a preferred embodiment, the agent is cyclopamine.

[0030] Yet another embodiment provides a method of treating a patientwith liver disease. A nestin-positive pancreatic stem cell is isolatedfrom a pancreatic islet of a donor and transferred into the patient,where the stem cell differentiates into a hepatocyte.

[0031] In a related embodiment, the stem cell is expanded ex vivo to aprogenitor cell, which is transferred into the patient and furtherdifferentiates into a hepatocyte.

[0032] In another related embodiment, the stem cell is differentiated exvivo to a progenitor cell, which is transferred into the patient andfurther differentiates into a hepatocyte. In another related embodiment,the stem cell is differentiated ex vivo into hepatocytes, which aretransplanted into the patient.

[0033] In these embodiments, the patient can also serve as the donor ofthe pancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

[0034] Yet another embodiment provides an isolated, nestin-positivehuman liver stem cell. In versions of this embodiment, the stem cell isimmunoprivileged. In versions of this embodiment, the stem cell does notexpress class I MHC antigens. In versions of this embodiment, the stemcell does not express class II MHC antigens. In versions of thisembodiment, the stem cell does not express class I or class II MHCantigens.

[0035] Yet another embodiment provides an isolated, nestin-positivehuman stem cell that is not a neural stem cell, that is capable oftransplant into an animal without causing graft versus host rejection.In versions of this embodiment, the stem cell is not majorhistocompatibility complex class I or class I restricted.

[0036] A “stem cell” as used herein is a undifferentiated cell which iscapable of essentially unlimited propagation either in vivo or ex vivoand capable of differentiation to other cell types. This can be tocertain differentiated, committed, immature, progenitor, or mature celltypes present in the tissue from which it was isolated, or dramaticallydifferentiated cell types, such as for example the erythrocytes andlymphocytes that derive from a common precursor cell, or even to celltypes at any stage in a tissue completely different from the tissue fromwhich the stem cell is obtained. For example, blood stem cells maybecome brain cells or liver cells, neural stem cells can become bloodcells, such that stem cells are pluripotential, and given theappropriate signals from their environment, they can differentiate intoany tissue in the body.

[0037] In one embodiment, a “stem cell” according to the invention isimmunologically blinded or immunoprivileged. As used herein,“immunologically blinded” or “immunoprivileged” refers to a cell thatdoes not elicit an immune response. As used herein, an “immune response”refers to a response made by the immune system to a foreign substance.An immune response, as used herein, includes but is not limited totransplant or graft rejection, antibody production, inflammation, andthe response of antigen specific lymphocytes to antigen. An immuneresponse is detected, for example, by determining if transplantedmaterial has been successfully engrafted or rejected, according tomethods well-known in the art, and as defined herein in the sectionentitled, “Analysis of Graft Rejection”. In one embodiment, an“immunogically blinded stem cell” or an “immunoprivileged stem cell”according to the invention can be allografted or xenografted withouttransplant rejection, and is recognized as self in the transplantrecipient or host.

[0038] Transplanted or grafted material can be rejected by the immunesystem of the transplant recipient or host unless the host isimmunotolerant to the transplanted material or unless immunosupressivedrugs are used to prevent rejection.

[0039] As used herein, a host that is “immunotolerant”, according to theinvention, fails to mount an immune response, as defined herein. In oneembodiment, a host that is “immunotolerant” does not reject or destroytransplanted material. In one embodiment, a host that is“immunotolerant” does not respond to an antigen by producing antibodiescapable of binding to the antigen.

[0040] As used herein, “rejection” refers to rejection of transplantedmaterial by the immune system of the host. In one embodiment, “rejectionmeans an occurrence of more than 90% cell or tissue necrosis of thetransplanted material in response to the immune response of the host. Inanother embodiment, “rejection” means a decrease in the viability suchthat the viability of the transplanted material is decreased by 90% ormore as compared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.As used herein, “graft versus host rejection” or “graft versus hostresponse” refers to a cell-mediated reaction in which T-cells of thetransplanted material react with antigens of the host.

[0041] As used herein, “host versus graft rejection” or “host versusgraft response” refers to a cell-mediated reaction in which cells of thehost's immune system attack the foreign grafted or transplantedmaterial.

[0042] In another embodiment of the invention, an immune response hasoccurred if production of a specific antibody (for example an antibodythat binds specifically to an antigen on the transplanted material, oran antibody that binds specifically to the foreign substance or aproduct of the foreign substance) is detected by immunological methodswell-known in the art, including but not limited to ELISA,immunostaining, immunoprecipitation and Western Blot analysis.

[0043] Stem cells express morphogenic or growth hormone receptors on thecell surface, and can sense, for example, injury-related factors thenlocalize to and take residence at sites of tissue injury, or sense theirlocal microenvironment and differentiate into the appropriate cell type.“Essentially unlimited propagation” can be determined, for example, bythe ability of an isolated stem cell to be propagated through at least50, preferably 100, and even up to 200 or more cell divisions in a cellculture system. Stem cells can be “totipotent,” meaning that they cangive rise to all the cells of an organism as for germ cells. Stem cellscan also be “pluripotent,” meaning that they can give rise to manydifferent cell types, but not all the cells of an organism. When a stemcell differentiates it generally gives rise to a more adult cell type,which may be a partially differentiated cell such as a progenitor cell,a differentiated cell, or a terminally differentiated cell. Stem cellscan be highly motile. “Nestin” refers to an intermediate filamentprotein having a sequence disclosed in Genbank Access No. X65964 (FIG.7).

[0044] A “pancreatic” stem cell means a stem cell that has been isolatedfrom pancreatic tissue and/or a cell that has all of the characteristicsof: nestin -positive staining, nestin gene expression, cytokeratin-19negative staining, long-term proliferation in culture, and the abilityto differentiate into pseudo-islets in culture.

[0045] A “liver” stem cell means a stem cell that has been isolated fromliver tissue and/or a cell that has all of the characteristics of:nestin -positive staining, nestin gene expression, and long-termproliferation in culture.

[0046] A “progenitor cell” is a cell that is derived from a stem cell bydifferentiation and is capable of further differentiation to more maturecell types.

[0047] As used herein, the term “insulin-producing beta cell” refers toany cell which can produce and secrete insulin in a similar amount tothat produced and secreted by a beta cell of the islets of Langerhans inthe human pancreas. Preferably, the secretion of insulin by aninsulin-producing beta cell is also regulated in a similar fashion tothe regulation of insulin secretion by a human beta cell in situ; forexample, insulin secretion should be stimulated by an increase in theglucose concentration in the solution surrounding the insulin-producingbeta cell.

[0048] “Pseudo-islet like” aggregates are artificial aggregates ofinsulin-secreting cells which resemble in form and function the isletsof Langerhans of the pancreas. Pseudo-islet like aggregates are createdex vivo under cell culture conditions. They are approximately 50-150 μmin diameter (compared to an average diameter of 100 μm for in situislets) and spheroid in form.

[0049] “Isolating” a stem cell refers to the process of removing a stemcell from a tissue sample and separating away other cells which are notstem cells of the tissue. An isolated stem cell will be generally freefrom contamination by other cell types and will generally have thecapability of propagation and differentiation to produce mature cells ofthe tissue from which it was isolated. However, when dealing with acollection of stem cells, e.g., a culture of stem cells, it isunderstood that it is practically impossible to obtain a collection ofstem cells which is 100% pure. Therefore, an isolated stem cell canexist in the presence of a small fraction of other cell types which donot interfere with the utilization of the stem cell for analysis orproduction of other, differentiated cell types. Isolated stem cells willgenerally be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%,or 99% pure. Preferably, isolated stem cells according to the inventionwill be at least 98% or at least 99% pure.

[0050] A stem cell is “expanded” when it is propagated in culture andgives rise by cell division to other stem cells and/or progenitor cells.Expansion of stem cells may occur spontaneously as stem cellsproliferate in a culture or it may require certain growth conditions,such as a minimum cell density, cell confluence on the culture vesselsurface, or the addition of chemical factors such as growth factors,differentiation factors, or signaling factors.

[0051] A stem cell, progenitor cell, or differentiated cell is“transplanted” or “introduced” into a mammal when it is transferred froma culture vessel into a patient. Transplantation, as used herein, caninclude the steps of isolating a stem cell according to the inventionand transferring the stem cell into a mammal or a patient.Transplantation can involve transferring a stem cell into a mammal or apatient by injection of a cell suspension into the mammal or patient,surgical implantation of a cell mass into a tissue or organ of themammal or patient, or perfusion of a tissue or organ with a cellsuspension. The route of transferring the stem cell or transplantation,will be determined by the need for the cell to reside in a particulartissue or organ and by the ability of the cell to find and be retainedby the desired target tissue or organ. In the case where a transplantedcell is to reside in a particular location, it can be surgically placedinto a tissue or organ or simply injected into the bloodstream if thecell has the capability to migrate to the desired target organ.

[0052] Transplantation, as used herein, can include the steps ofisolating a stem cell according to the invention, and culturing andtransferring the stem cell into a mammal or a patient. Transplantation,as used herein, can include the steps of isolating a stem cell accordingto the invention, differentiating the stem cell, and transferring thestem cell into a mammal or a patient. Transplantation, as used herein,can include the steps of isolating a stem cell according to theinvention, differentiating and expanding the stem cell and transferringthe stem cell into a mammal or a patient.

[0053] Treatment with an immunosuppressive agent can be accomplished byadministering to a patient in need thereof any agent which prevents,delays the occurrence of or decreases the intensity of the desiredimmune response, e.g., rejection of a transplanted cell, tissue, ororgan.

[0054] As used herein, “immunosuppression” refers to prevention of theimmune response (for example by the administration of an“immunosuppresive agent”, as defined herein) such that an “immuneresponse”, as defined herein, is not detectable. As used herein,“prevention” of an immune response means an immune response is notdetectable. An immune response (for example, transplant rejection orantibody production) is detected according to methods well-known in theart and defined herein.

[0055] “Immunosuppression” according to the invention also means a delayin the occurrence of the immune response as compared to any one of atransplant recipient that has not received an immunosuppresive agent, ora transplant recipient that has been transplanted with material that isnot “immunologically blinded” or “immunoprivileged”, as defined herein.A delay in the occurrence of an immune response can be a short delay,for example 1hr-10 days, i.e., 1 hr, 2, 5 or 10 days. A delay in theoccurrence of an immune response can also be a long delay, for example,10 days-10 years (i.e., 30 days, 60 days, 90 days, 180 days, 1, 2, 5 or10 years).

[0056] “Immunosuppression” according to the invention also means adecrease in the intensity of an immune response. According to theinvention, the intensity of an immune response can be decreased suchthat it is 5-100%, preferably, 25-100% and most preferably 75-100% lessthan the intensity of the immune response of any one of a transplantrecipient that has not received an immunosuppresive agent, or atransplant recipient that has been transplanted with material that isnot “immunologically blinded” or “immunoprivileged”, as defined herein.The intensity of an immune response can be measured by determining thetime point at which transplanted material is rejected. For example, animmune response comprising rejection of transplanted material at day 1,post-transplantation, is of a greater intensity than an immune responsecomprising the rejection of transplanted material at day 30,post-transplantation. The intensity of an immune response can also bemeasured by quantitating the amount of a particular antibody capable ofbinding to the transplanted material, wherein the level of antibodyproduction correlates directly with the intensity of the immuneresponse. Alternatively, the intensity of an immune response can bemeasured by determining the time point at which a particular antibodycapable of binding to the transplanted material is detected.

[0057] Various strategies and agents can be utilized forimmunosuppression. For example, the proliferation and activity oflymphocytes can be inhibited generally with agents such as, for example,FK-506, or cyclosporin or other immunosuppressive agents. Anotherpossible strategy is to administer an antibody, such as an anti-GAD65monoclonal antibody, or another compound which masks a surface antigenon a transplanted cell and therefore renders the cell practicallyinvisible to the immune system of the host.

[0058] An “immunosuppressive agent” is any agent that prevents, delaysthe occurrence of or reduces the intensity of an immune reaction againsta foreign cell in a host, particularly a transplanted cell. Preferredare immunosuppressive agents which suppress cell-mediated immuneresponses against cells identified by the immune system as non-self.Examples of immunosuppressive agents include but are not limited tocyclosporin, cyclophosphamide, prednisone, dexamethasone, methotrexate,azathioprine, mycophenolate, thalidomide, FK-506, systemic steroids, aswell as a broad range of antibodies, receptor agonists, receptorantagonists, and other such agents as known to one skilled in the art.

[0059] A “mitogen” is any agent that stimulates mitosis and cellproliferation of a cell to which the agent is applied.

[0060] A “differentiation factor” is any agent that causes a stem cellor progenitor cell to differentiate into another cell type.Differentiation is usually accomplished by altering the expression ofone or more genes of the stem cell or progenitor cell and results in thecell altering its structure and function.

[0061] A “signaling factor” as used herein is an agent secreted by acell which has an effect on the same or a different cells. For example,a signaling factor can inhibit or induce the growth, proliferation, ordifferentiation of itself, neighboring cells, or cells at distantlocations in the organism. Signaling factors can, for example, transmitpositional information in a tissue, mediate pattern formation, or affectthe size, shape and function of various anatomical structures.

[0062] As used herein, a mammal refers to any mammal including but notlimited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster,horse, cow or pig.

[0063] A “non-human mammal”, as used herein, refers to any mammal thatis not a human.

[0064] As used herein, “allogeneic” refers to genetically differentmembers of the same species.

[0065] As used herein, “isogeneic” refers to of an identical geneticconstitution.

[0066] As used herein, “xenogeneic” refers to members of a differentspecies.

[0067] As used herein, “culturing” refers to propagating or nurturing acell, collection of cells, tissue, or organ, by incubating for a periodof time in an environment and under conditions which support cellviability or propagation. Culturing can include one or more of the stepsof expanding and proliferating a cell, collection of cells, tissue, ororgan according to the invention.

[0068] The invention also provides for a pharmaceutical compositioncomprising the isolated stem cells of the invention admixed with aphysiologically compatible carrier.

BRIEF DESCRIPTION OF TILE DRAWINGS

[0069]FIGS. 1A and 1B show dual fluorescence immunocytochemical stainingof rat pancreatic islets at embryonic day 16 (FIG. 1A) and at day 60after birth (FIG. 1B). Immunostaining with an antibody for nestin isshown in white (red in the original, with Cy3 as fluorophore) and withan antibody for insulin is shown in grey (green in the original, withCy2 as fluorophore).

[0070]FIG. 2 shows the result of RT-PCR performed using mRNA obtainedfrom 50 rat islets. Forward and reverse primers are indicated. Thesingle band of 834 bp was sequenced and identified substantially as thesequence for nestin.

[0071]FIG. 3 shows nestin-positive cells that have proliferated out froma cultured rat islet.

[0072]FIG. 4 shows the development of islet like structures in culture.

[0073]FIG. 5 shows the results of RT-PCR analysis of islet-likestructures generated in culture. Expression of NCAM and cytokeratin-19(CK19) was detected.

[0074]FIG. 6 shows the stimulation of nestin mRNA expression by highglucose. APRT was examined as a control.

[0075]FIG. 7 is the amino acid and nucleotide sequences of nestin.

[0076]FIG. 8 depicts expression of the neural stem cell-specific markernestin in a distinct cell population within pancreatic islets asdetermined by immunocytochemistry or RT-PCR.

[0077]FIG. 9 depicts characterization of nestin in stem cells isolatedfrom the pancreas by inumunocytochemistry and RT-PCR.

[0078]FIG. 10 depicts expression of homeodomain protein IDX-1 andproglucagon in human islet-like clusters derived from nestin-positiveislet progenitor cells (NIPs).

[0079]FIG. 11 demonstrates localization of nestin-positive cells tolocalized regions of the ducts of the rat pancreas.

[0080]FIG. 12 depicts alternative models for the origin of pancreaticduct cells that are progenitors of islet endocrine cells.

[0081]FIG. 13A and B depicts immunofluorescent staining of nestinpositive liver stem cells.

[0082]FIG. 14 depicts the sequential appearance of transcription factorsduring development of the murine endocrine pancreas.

[0083]FIG. 15 depicts expression of neuroendocrine, exocrine pancreaticand hepatic markers in human NIP cultures containing stem cells.

[0084]FIG. 16 depicts expression of proglucagon and insulin mRNA asdetermined by RT-PCR and insulin secretion.

DETAILED DESCRIPTION OF THE INVENTION

[0085] The present inventors have identified and isolated a specialsubclass of ductal cells from the islets of Langerhans of mammalianpancreas that have the functional and molecular characteristics of stemcells. In particular, these newly discovered pancreatic stem cells arecharacterized inter alia by one or more (and preferably all of):nestin-positive staining, nestin gene expression, cytokeratin-19negative staining, long-term proliferation in culture, and the abilityto differentiate into pseudo-islets in culture. The present inventorshave also identified liver cells that exhibit nestin-positive staining.

[0086] In one embodiment, the invention provides stem cells for avariety of applications, including but not limited to cellularreplacement therapy for type I insulin-dependent diabetes and otherforms of diabetes as well as the development of research tools to studythe onset and progression of various diabetic conditions, hormonalabnormalities, and genetic diseases or conditions, such as theassociation of polymorphisms with particular physiologic or pathologicstates. The stem cells of the invention can also be used to carry outgene therapy of endocrine pancreatic or other tissues in isograft,allograft or xenograft transplantations. Further, the stem cellsdescribed herein can be used to produce recombinant cells, artificialtissues, and replacement organs in culture. They can also be used forthe ex vivo production of insulin and other hormones. Molecularcharacteristics of pancreatic stem cells discovered by the inventors,such as nestin-positive and cytokeratin-19 negative staining, or liverstem cells, such as nestin-positive staining, can be used in variousdiagnostic, pathological, or investigative procedures to identify,localize, and quantitate stem cells in tissues from a patient orexperimental animal.

[0087] Identification of Stem Cells in Pancreatic Islets

[0088] Previous investigators have focused on ductal epithelial cells ofpancreatic islets or exocrine tissue as a possible source of stem cellsfor the neogenesis of islet endocrine cells. Nestin is an intermediatefilament protein that was cloned by screening a CDNA library from E 15rat embryos with a monoclonal antibody named R.401 (Hockfield & McKay,1985; Lendahl et al., 1990). Nestin was primarily found inneuroepithelial stem cells and is expressed in the developing centralnervous system. After maximum levels are reached in the rat embryo atE16, nestin expression declines to almost undetectable levels in adultcerebral cortex, coinciding with terminal differentiation of earlynestin-expressing progenitor cells (Lendahl et al., 1990). Nestin wasinitially found exclusively in stem cells of the embryonic developingbrain and skeletal muscle (Lendahl et al., 1990). Later studiesidentified nestin-positive neural stem cells in the subependymal layerof the adult mammalian forebrain (Morshead et al., 1994).Nestin-positive stem cells have been shown to be pluripotential evenwhen isolated from adult mice or rat brain. For example, nestin-positivestem cells can generate all three major classes of neural cells inculture: neurons, astrocytes, and oligodendrocytes (Reynolds & Weiss,1996). Nestin-positive neural stem cells respond to spinal cord injuryby proliferation and degeneration of migratory cells that differentiateinto astrocytes, participate in scar formation (Johansson et al., 1999)and restore hematopoietic cells of the bone marrow after infusion intoirradiated mice (Bjornson et al., 1999).

[0089] Characterization of Stem Cells

[0090] Stem cells according to the invention can be identified by theirexpression of nestin by, for example, FACS, immunocytochemical staining,RT-PCR, Southern Northern and Western blot analysis, and other suchtechniques of cellular identification as known to one skilled in theart.

[0091] Immunocytochemical staining, for example, is carried outaccording to the following method. Cryosections (6 μM) prepared frompancreata or liver, as well as cells, are fixed with 4% paraformaldehydein phosphate. Cells are first blocked with 3% normal donkey serum for 30min at room temperature and incubated with a primary antisera to theprotein of interest overnight at 4° C. The antisera is rinsed off withPBS and incubated with the appropriate fluorescently labeled secondaryantisera for 1 hour at room temperature. Slides are then washed with PBSand coverslipped with fluorescent mounting medium (Kirkegaard and PerryLabs, Gaithersburg, Md.). Fluorescence images are obtained using a ZeissEpifluorescence microscope equipped with an Optronics TEC-470 CCD camera(Optronics Engineering, Goleta, Calif.) interfaced with a PowerMac 7100installed with IP Lab Spectrum analysis software (Signal Analytics Corp,Vienna, Va.).

[0092] Antisera useful according to the invention include the following:mouse monoclonal antibodies to human cytokeratin 19 (clone K4.62, Sigma,St. Louis, Mo.), rabbit polyclonal antisera to rat nestin and to IDX-1(prepared by immunizations of rabbits with a purified GST-nestin fusionprotein or the last twelve amino acids of rat IDX-1, respectively)(McManus et al., 1999, J. Neurosci., 19:9004-9015), guinea piganti-insulin and anti-pancreatic polypeptide antisera, obtained fromLinco, St. Charles, Mo., and mouse antiglucagon and rabbitantisomatostatin antisera, purchased from Sigma (St. Louis, Mo.) andDAKO (Carpinteria, Calif.), respectively, mouse anti-human galanin(Peninsula Laboratories, Belmont, Calif.), collagen IV antisera (CaltagLaboratories, San Francisco, Calif.), mouse anti-rat MHC class I serum(Seroteck), and antirat MHC class II serum. The invention contemplatesthat other antisera directed to such markers is available, or will bedeveloped. Such other antisera is considered to be within the scope ofthe invention.

[0093] RT-PCR and Southern blot analysis are performed according to thefollowing methods. Total cellular RNA prepared from rat or human isletsis reverse transcribed and amplified by PCR for about 35 cyclesdepending on the desired degree of amplification, as describedpreviously (Daniel, et al., 1998, Endocrinology, 139:3721-3729).Oligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are: Rat nestin: Forward,5′gcggggcggtgcgtgactac3′; Reverse, 5′aggcaagggggaagagaaggatgt3′;Hybridization, 5′aagctgaagccgaatttccttgggataccagagga3′. Rat Forward,5′acagccagtacttcaagacc3′; keratin 19: Reverse, 5′ctgtgtcagcacgcacgtta3′;Hybridization, 5′tggattccacaccaggcattgaccatgcca3′. Rat NCAM: Forward,5′cagcgttggagagtccaaat3′; Reverse, 5′ttaaactcctgtggggttgg3′;Hybridization, 5′aaaccagcagcggatctcagtggtgtggaacgatgat3′. Rat IDX-1Forward, 5′atcactggagcagggaagt3′ Reverse, 5′gctactacgtttcttatct3′Hybridization, 5′gcgtggaaaagccagtggg3′ Human Forward,5′agaggggaattcctggag3′; nestin: Reverse, 5′ctgaggaccaggactctcta3′;Hybridization, 5′tatgaacgggctggagcagtctgaggaaagt3′. Human Forward,5′cttttcgcgcgcccagcatt3′; keratin: Reverse, 5′gatcttcctgtccctcgagc3′;Hybridization, 5′aaccatgaggaggaaatcagtacgctgagg3′. Human Forward,5′atctggactccaggcgtgcc3′; glucagon: Reverse, 5′agcaatgaattccttggcag3′;Hybridization, 5′cacgatgaatttgagagacatgctgaaggg3′; Human Forward, 5′agaacagcacgtacacagcc 3′ E-Cadherin Reverse, 5′cctccgaagaaacagcaaga 3′Hybridization, 5′ tctcccttcacagcagaactaacacacggg 3′ Human Forward, 5′gcagtcctgccatcaatgtg 3′ transthyretin Reverse, 5′ gttggctgtgaataccacct3′ Hybridization, 5′ctggagagctgcatgggctcacaactgagg 3′ Human Forward,5′gactttccagcagtcccata 3′ Pancreatic Reverse, 5′ gtttacttcctgcagggaac 3′Amylase Hybridization, 5′ ttgcactggagaaggattacgtggcgttcta 3′ HumanForward, 5′ tgaaggcgagaaggtgttcc 3′ procarboxy- Reverse, 5′ttcgagatacaggcagatat 3′ peptidase Hybridization, 5′agttagacttttatgtcctgcctgtgctca 3′ Human Forward, 5′ cttcaggctgcaccaagtgt3′ Synapto- Reverse, 5′ gttgaccatagtcaggctgg 3′ physin Hybridization, 5′gtcagatgtgaagatggccacagacccaga 3′ Human Forward, 5′ gcatcaaatgtcagccctgg3′ Hepatocyte Reverse, 5′ caacgctgacatggaattcc 3′ Growth Hybridization,5′ tcgaggtctcatggatcatacagaatcagg 3′ Factor (HGF) Human Forward, 5′caatgtgagatgtctccagc 3′ cMET Reverse, 5′ ccttgtagattgcaggcaga 3′ (HGF-Hybridization, 5′ ggactcccatccagtgtctccagaagtgat 3′ receptor) HumanForward, 5′gagtagcagctcagactgcc 3′ XBP-1 Reverse, 5′gtagacctctgggagctcct 3′ Hybridization, 5′ cgcagcactcagactacgtgcacctctgca3′ Human Forward, 5′ gcagctgctcaactaatcac 3′ Glut-2 Reverse, 5′tcagcagcacaagtcccact 3′ Hybridization, 5′ acgggcattcttattagtcagattattggt3′ Human Forward, 5′ aggcttcttctacaca3′ Insulin Reverse, 5′caggctgcctgcacca 3′ Hybridization, 5′ aggcagaggacctgca 3′

[0094] Other such sequences are possible and such sequences areconsidered to be within the scope of the art. As a general guide,primers are selected from two different exons and encompass at least oneintronic sequence. In addition, an RT minus control is run for mostsamples. PCR amplification is effectuated at 94° C. for 1 min followedby 94° C. for 10 secs, 58/56° C. for 10 secs, 72° C. for 1 min, 35cycles, and 72° C. for 2 min. The annealing temperature is 58° C. forrat nestin and 56° C. for the remaining primer pairs.

[0095] For RT-PCR of mRNA isolated from a mammal that is not rat orhuman, oligonucleotides that are specific for the amplified nucleic acidfrom the mammalian species being analyzed are prepared. The selectionand use of such primers is known to one skilled in the art.

[0096] For Southern hybridization oligonucleotide probes are labeledwith an appropriate radionuclide, such as γ³²P ATP, using conventionaltechniques. Radiolabeled probes are hybridized to PCR productstransferred to nylon membranes at 37° C. for one hour, then washed in1×SSC+0.5% SDS at 55° C. for 10-20 min or 0.5×SCC+0.5% SDS at 42° forthe human PCR products.

[0097] Nestin as a Marker of Pancreatic Stem Cells

[0098] The inventors have now unexpectedly discovered that the pancreasof adult mammals, including humans, contains cells that express nestin.Importantly, the distribution of nestin-positive cells in the pancreasdoes not correspond to the distribution of hormone-producing cells. Forexample, fluorescently labeled antibodies specifically reactive toinsulin or glucagon label the beta and alpha cells of the islets,respectively, whereas the inventors have observed that in mice, rats,and humans, fluorescently labeled nestin antibodies localize only tocertain cells of the ductular epithelium and not to alpha, beta, delta,and pancreatic peptide producing cells in pancreatic islets (FIG. 1).The inventors also observed that antibodies specific for collagen IV, amarker for vascular endothelial cells, galanin, a marker of nerveendings, and cytokeratin 19, a marker for ductal cells, do notcolocalize with nestin antibodies. Furthermore, the inventors have foundthat nestin-positive islet cells do not co-label with antibodies forinsulin, glucagon, somatostatin, or pancreatic polypeptide (FIG. 1).This suggests that these nestin-containing cells are not endocrinecells, ductal cells, neural cells, or vascular endothelial cells, butmay represent a truly distinct cell type within the islet that has notpreviously been described. The inventors have found nestin-positivecells in the islets, as well as localized regions of the pancreaticducts, and within centroacinar regions of the exocrine pancreas.

[0099] The expression of nestin mRNA in isolated islets was detectedusing RT-PCR with RNA from isolated rat islets (FIG. 2). The functionalproperties of nestin-positive pancreatic cells were investigated usingcell culture techniques and by the isolation of nestin-positive cellsfrom islets, both of which are described below.

[0100] The inventors have also discovered that the liver of ratscontains cells that express nestin (FIG. 13).

[0101] Cytokeratin-19 as a Marker for a Distinct Population of DuctEpithelial Cells

[0102] Cytokeratin-19 (CK-19) is another intermediate filament protein.CK-19 and related cytokeratins have previously been found to beexpressed in pancreatic ductal cells (Bouwens et al., 1994). Theinventors have discovered, however, that while CK-19 expression isindeed confined to the ductules, fluorescent antibodies specific forCK-19 label distinct ductal cells from those labeled withnestin-specific antibodies. This suggests that nestin-positive cells inislets may be a distinct cell type of ductal cell from CK-19 positivecells.

[0103] Isolated Stem Cells from Pancreatic Islets and Their Use

[0104] Stem cells can be isolated from a preparation of pancreatictissue, for example, islets obtained from a biopsy sample of tissue froma diabetic patient. The stem cells can then be expanded ex vivo and theresulting cells transplanted back into the donor as an isograft. Insidethe donor, they may differentiate to provide insulin-secreting cellssuch as beta cells to replace beta cells lost to the autoimmune attackwhich caused the diabetes. This approach can overcome the problems ofimmune rejection resulting from transplantation of tissue, for example,islets from another individual who might serve as the donor. In oneembodiment of the invention, the use of isografted stem cells allowsanother technique to be performed in an effort to avoid the immunerejection, namely genetic therapy of the transplanted cells to renderthem resistant to immune attack, such as the autoimmunity present inindividuals with type 1 diabetes. A further advantage of using stemcells over whole islets is that transplanted stem cells candifferentiate in situ and better adapt to the host environment, forexample, providing appropriate microcirculation and a complement ofdifferent islet cell types which responds to the physiological needs ofthe host. Another embodiment of the invention contemplates the use ofpartially differentiated stem cells ex vivo, for example, to formprogenitor cells, which are subsequently transplanted into the host,with further differentiation optionally taking place within the host.Although the use of an isograft of stem cells, progenitor cells, orpseudo-islets is preferred, another embodiment contemplates the use ofan allograft of stem cells, progenitor cells, or pseudo-islets obtainedfrom another individual or from a mammal of another species.

[0105] In yet another embodiment of the invention, the stem cells areimmunologically blind or immunoprivileged. In one embodiment of thisaspect of the invention, immunoprivileged stem cells do not expresssufficient amounts of class I and/or class II major histocompatibilityantigens (a.k.a. HLA or human leukocyte antigen) to elicit an immuneresponse from the host. For example, these stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

[0106] In another embodiment of this aspect of the invention,immunoprivileged stem cells do not express class I MHC antigens and/orclass II MHC antigens. These stem cells, obtained from allogeneic orxenogeneic sources do not initiate a host versus graft response inimmunocompetent transplant recipients.

[0107] In another embodiment of the invention, human tissue graftscomprising stem cells express both human specific class I and class IIMHC antigens, but are recognized by immunocompetent mice as self, and donot undergo host versus graft rejection. These stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

[0108] The invention also provides for methods of isolating stem cellsfrom a xenogenic donor, and transplanting the resulting cells into amammal of another species (e.g. murine stem cells are transplanted intoa human, for example, a diabetic human patient) as a xenograft.

[0109] The invention provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of nestin-positive stem cellswherein the stem cells are cultured for a period of time, for example,2-4 hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation.

[0110] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of nestin-positive stem cellswherein the stem cell is expanded for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation togive rise by cell division to other stem cells or progenitor cells.

[0111] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of stem cells wherein thenestin-positive stem cells are induced to differentiate into aprogenitor cell by treatment with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleicacid molecule encoding IDX-l, GLP-1, exendin-4, betacellulin, activin A,TGF-P, and combinations thereof for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation. Inthe case of a pancreatic stem cell, the stem cell subsequentlydifferentiates into a pancreatic progenitor cell.

[0112] The invention provides for methods of performing isogeneic,allogeneic or xenogeneic transplants wherein nestin-positive stem cellsare not cultured, expanded or differentiated prior to transplantation orwherein nestin-positive stem cells are cultured and/or expanded and/ordifferentiated prior to transplantation.

[0113] Nestin-positive cells can be proliferated in culture fromisolated pancreatic islets and subsequently isolated to form a stem cellline capable of essentially unlimited propagation.

[0114] The inventors discovered that nestin expressing cells grow out ofcultured islets and can be observed growing around the islets as earlyas about four days in culture. These cells have a neuron-like morphology(FIG. 3), show nestin-positive staining, and express nestin mRNA. Isletscontaining nestin-positive cells can be separated from other cells,e.g., fibroblasts, that proliferate from the cultured islets by exposinga suspension of the islets to concanavalin A. The islets containingnestin-positive stem cells will not adhere to a concanavalin A coatedculture vessel, for example, allowing the islets to be simply decantedwhile other cell types remain attached to the vessel. The islets arethen plated on wells that do not have a concanavalin A coating, wherethey adhere. The details of the culture and isolation procedure aredescribed for rat cells in Example 1 below. Similar results have beenobtained with human cells.

[0115] Formation of Pseudo-Islets and Ductal Structures in Culture

[0116] One embodiment of the invention provides an alternative totransplantation of stem cells or progenitor cells by causing them toform pseudo-islet like aggregates that can be transplanted into apatient with insufficient islet cell mass to maintain physiologicalcontrol without hormone therapy. Islet-derived stem cells can beprepared from cultured islets as indicated above or obtained from apropagating stem cell line. The stem cells can then be induced todifferentiate by exposing them to various growth factors. This processis illustrated in Examples 2 and 3.

[0117] Differentiation of Stem Cells or Progenitor Cells to Islet Cells

[0118] Growth factors that may induce differentiation of pancreatic stemcells include but are not limited to EGF-2, basic FGF, high glucose,KGF, HGF/SF, GLP-1, exendin-4, betacellulin, activin A, TGF-β, andcombinations thereof. GLP-1 refers to glucagon-like peptide-1. Highglucose refers to a higher glucose concentration than the concentrationnormally used in culturing the stem cells. For example, the stem cellscan be normally cultured and propagated in about 5.6 mM glucose, and thehigh glucose concentration refers to another concentration above 5.6 mM.In the preferred embodiment, a concentration of 16.7 mM is contemplated.In Example 2, one possible growth factor treatment is described usingbasic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

[0119] In addition to growth factors added to the medium of culturedcells, further growth factors can contribute to differentiation whenstem cells are implanted into an animal or a human. In that situation,many growth factors which are either known or unknown may be secreted byendogenous cells and exposed to the stem cells in situ. Implanted stemcells can be induced to differentiate by any combination of endogenousand exogenously administered growth factors that are compatible with thedesired outcome, i.e., the final differentiated cell type or types andthe anatomical structures (e.g., tissues or organs) formed.

[0120] One embodiment provides an approach to stimulatingdifferentiation, that is to administer downstream effectors of growthfactors or to transfect stem cells or progenitor cells with a nucleicacid molecule encoding such effectors. One example is IDX-1, which is atranscription factor induced by GLP-1 or exendin-4. Introducingeffectors such as IDX-1 can trigger differentiation to form endocrineislet cells.

[0121] Analysis of Graft Rejection

[0122] The invention provides for an in vivo procedure for evaluatingthe survival of transplanted material. Experimental transplant rejectionis analyzed by transplanting an immunosuppressed or anon-immunosuppressed mammal, with a stem cell or a pseudo-islet likeaggregate according to the invention.

[0123] For example, non-immunosuppressed C57BL/6 mice are transplanted(for example, under the renal capsules) with human stem cells accordingto the invention. Graft rejection is analyzed by sacrificing thetransplant recipient and staining for viability, or performingimmunocytochemical staining at the site of the grafted material (i.e.,an organ or tissue present at the site of the grafted material) at asuitable post-transplantation time point. The time point at whichstaining (for example hematoxylin/eosin or immunostaining) of the siteof the grafted material is made can vary, for example, according to theaverage survival time, or the expected survival time of a transplantedmammal. The site of the graft is analyzed, for example by staining, 1day to 10 years (i.e., 1, 5, 10, 30, 100 or more days, 1, 2, 5, or 10years) post-transplantation, preferably 10 days to 1 yearpost-transplantation and most preferably, 10-100 dayspost-transplantation. For example, if transplanted material isintroduced under the renal capsule of a mouse, the kidney of thetransplanted mouse is inspected. Transplanted material is successfullyengrafted (i.e., not rejected) if, the transplanted material is stilldetectable and/or the transplanted material has proliferated into atissue mass.

[0124] Detection of the transplanted material and proliferation of thetransplanted material is determined, for example, by hematoxylin/eosinstaining of a frozen section prepared from the transplant site (i.e.,the kidney) and the detection of new growth that is not derived from thetransplant recipient (i.e., not host kidney derived). In the case ofxenogeneic transplantation, transplanted material is successfullyengrafted if specific immunostaining with antisera specific for anantigen from the species from which the transplanted material isderived, according to methods of immunocytochemical staining known inthe art and described herein, identifies positive cells. Alternatively,in embodiments wherein a xenogeneic transplantation is performed,transplanted material is successfully engrafted if molecules (i.e., aprotein or an antigen) derived from the transplant species (that is thespecies from which the transplanted material is derived) are detected inthe blood of the transplant recipient.

[0125] As used herein, “rejection” refers to rejection of transplantedmaterial by the immune system of the host. In one embodiment, “rejectionmeans an occurrence of more than 90% cell or tissue necrosis of thetransplanted material in response to the immune response of the host. Inanother embodiment, “rejection” means a decrease in the viability suchthat the viability of the transplanted material is decreased by 90% ormore as compared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.

[0126] Methods of Transplantation

[0127] The invention provides for methods of transplantation in to amammal. A stem cell, progenitor cell, or differentiated cell is“transplanted” or “introduced” into a mammal when it is transferred froma culture vessel into a patient.

[0128] Transplantation, according to the invention can include the stepsof isolating a stem cell according to the invention and transferring thestem cell into a mammal or a patient. Transplantation according to theinvention can involve transferring a stem cell into a mammal or apatient by injection of a cell suspension into the mammal or patient,surgical implantation of a cell mass into a tissue or organ of themammal or patient, or perfusion of a tissue or organ with a cellsuspension. The route of transferring the stem cell or transplantation,will be determined by the need for the cell to reside in a particulartissue or organ and by the ability of the cell to find and be retainedby the desired target tissue or organ. In the case where a transplantedcell is to reside in a particular location, it can be surgically placedinto a tissue or organ or simply injected into the bloodstream if thecell has the capability to migrate to the desired target organ.

[0129] Transplantation, according to the invention, can include thesteps of isolating a stem cell according to the invention, and culturingand transferring the stem cell into a mammal or a patient. In anotherembodiment, transplantation, as used herein, can include the steps ofisolating a stem cell according to the invention, differentiating thestem cell, and transferring the stem cell into a mammal or a patient.Transplantation, as used herein, can include the steps of isolating astem cell according to the invention, differentiating and expanding thestem cell and transferring the stem cell into a mammal or a patient.

[0130] Methods of Treating Insulin-Dependent Diabetes Using PancreaticStem Cells

[0131] Stem cells are useful to replace lost beta cells from Type 1diabetes patients or to increase the overall numbers of beta cells inType 2 diabetes patients. The diabetes patient will preferably serve asthe donor of pancreatic tissue used to produce stem cells, progenitorcells, or pseudo-islet like aggregates. Stem cells exist within theadult pancreatic islets as well as the pancreatic ducts. After adiabetic patient undergoes pancreatic biopsy, islets are isolated fromthe biopsy tissue and prepared for culture ex vivo preferably within 24hours. Stem cells can be proliferated and isolated by the methodsdescribed above within 2-3 weeks. Stem cells can be transplanted backinto the patient directly following isolation or after a period ofdifferentiation which is induced by growth factors. Islets can beproduced by subculture as described in Example 2. The whole process ofsurgical pancreas biopsy and transplantation can be performed within aperiod of about 30 days.

[0132] In one embodiment of the invention, pluripotential stem cells areused. These cells are immunologically blinded or immunoloprivileged,such that in allogeneic or xenogeneic transplants, they are recognizedas self by the recipient, and are not MHC restricted by class I or classII antigens. In one aspect of this embodiment of the invention, thesecells do not express MHC class I and/or class II antigens.

[0133] In another embodiment of the invention, the recipient of thetransplant may demonstrate host vs. graft rejection of othertransplanted cells, which can be combated by the administration ofblocking antibodies to, for example, an autoantigen such as GAD65, bythe administration of one or more immunosuppressive drugs describedherein, or by any method known in the art to prevent or ameliorateautoimmune rejection.

[0134] Alternatively, stem cells isolated from a non-human mammalaccording to the invention, are transplanted into a human diabetespatient. Prior to the transplantation step the stem cells may becultured, and/or expanded and/or differentiated.

[0135] Methods of Treating Patients Suffering from Liver Disease UsingPancreatic Stem Cells

[0136] The ability of pancreatic stem or progenitor cells totransdifferentiate to form hepatocytes is well known (Bisgaard &Thorgeirsson, 1991). The pancreatic stem cells of the present inventioncan be used to provide hepatocytes for a patient suffering from a liverdisease such as cirrosis, hepatitis, or hepatic cancer in which thefunctional mass of hepatic tissue has been reduced. The stem cells ofthe invention can also be treated by gene therapy to correct a geneticdefect and introduced into a patient to restore hepatic function.Nestin-positive stem cells can be differentiated either in culture or invivo by applying one or more growth factors, or other treatment such astransfection with a nucleic acid molecule, that results indifferentiation of the stem cells to hepatocytes. In one embodiment, theinvention contemplates the use of cyclopamine to suppress, for example,sonic hedgehog, resulting in hepatocyte formation. In another embodimentof the invention, the stem cells can be transplanted without any ex vivotreatment and the appropriate growth factors can be provided in situwithin the patient's body. In yet another embodiment, the stem calls canbe treated with growth factors or other agents ex vivo and subsequentlytransplanted into the patient in a partially differentiated orterminally differentiated state. Other aspects of the invention,including methods of transfecting stem cells or progenitor cells,dosages and routes of administration, pharmaceutical compositions,donor-isograft protocols, and immunosuppression methods, can bepracticed with transdifferentiation to hepatocytes just as for thedifferentiation to pancreatic tissues.

[0137] The invention specifically contemplates transplanting intopatients isogeneic, allogeneic, or xenogeneic stem cells, or anycombination thereof.

[0138] Methods of Stem Cell Transfection

[0139] A variety of methods are available for gene transfer intopancreatic stem cells. Calcium phosphate precipitated DNA has been usedbut provides a low efficiency of transformation, especially fornonadherent cells. In addition, calcium phosphate precipitated DNAmethods often result in insertion of multiple tandem repeats, increasingthe likelihood of disrupting gene function of either endogenous orexogenous DNA (Boggs, 1990). The use of cationic lipids, e.g., in theform of liposomes, is also an effective method of packaging DNA fortransfecting eukaryotic cells, and several commercial preparations ofcationic lipids are available. Electroporation provides improvedtransformation efficiency over the calcium phosphate protocol. It hasthe advantage of providing a single copy insert at a single site in thegenome. Direct microinjection of DNA into the nucleus of cells is yetanother method of gene transfer. It has been shown to provideefficiencies of nearly 100% for short-term transfection, and 20% forstable DNA integration. Microinjection bypasses the sometimesproblematic cellular transport of exogenous DNA through the cytoplasm.The protocol requires small volumes of materials. It allows for theintroduction of known amounts of DNA per cell. The ability to obtain avirtually pure population of stem cells would improve the feasibility ofthe microinjection approach to targeted gene modification of pancreaticstem cells. Microinjection is a tedious, highly specialized protocol,however. The very nature of the protocol limits the number of cells thatcan be injected at any given time, making its use in large scaleproduction limited. Gene insertion into pancreatic stem cells usingretroviral methods is the preferred method. Retroviruses provide arandom, single-copy, single-site insert at very high transfectionefficiencies. Other such transfection methods are known to one skilledin the art and are considered to be within the scope of this invention.

[0140] Retroviral Transformation Of Pancreatic Stem Cells

[0141] Gene transfer protocols for pancreatic cells can involveretroviral vectors as the “helper virus” (i.e., encapsidation-defectiveviral genomes which carry the foreign gene of interest but is unable toform complete viral particles). Other carriers such as DNA-mediatedtransfer, adenovirus, SV40, adeno-associated virus, and herpes simplexvirus vectors can also be employed. Several factors should be consideredwhen selecting the appropriate vector for infection. It is sometimespreferable to use a viral long terminal repeat or a strong internalpromoter to express the foreign gene rather than rely on splicedsubgenomic RNA.

[0142] The two primary methods of stem cell transformation areco-culture and supernatant infection. Supernatant infection involvesrepeated exposure of stem cells to the viral supernatant. Co-cultureinvolves the commingling of stem cells and an infected “package cellline” (see below) for periods of 24 to 48 hours. Co-culture is typicallymore efficient than supernatant infection for stem cell transformation.After co-culture, infected stem cells are often further cultured toestablish a long term culture (LTC).

[0143] The cell line containing the helper virus is referred to as thepackage cell line. A variety of package cell lines are currentlyavailable. An important feature of the package cell line is that it doesnot produce replication-competent helper virus.

[0144] In one embodiment of the invention animals or patients from whomstem cells are harvested may be treated with 5- fluorouracil (5-FU)prior to extraction. 5-FU treated stem cells are more susceptible toretroviral infection than untreated cells. 5-FU stem cells dramaticallyreduce the number of clonogenic progenitors, however.

[0145] In another embodiment, harvested stem cells may be exposed tovarious growth factors, such as those employed to promote proliferationor differentiation of pancreatic stem cells. Growth factors can beintroduced in culture before, during, or after infection to improve cellreplication and transduction. Studies report the use of growth factorsincrease transformation efficiency from 30 to 80%.

[0146] Typical Retroviral Transformation Protocol

[0147] The ex vivo transduction of mammalian pancreatic stem cells andsubsequent transplantation into nonablated recipients sufficient toobtain significant engraftment and gene expression in various tissuescontaining their progeny cells has been shown in mice. The target cellsare cultured for 2-4 days in the presence of a suitable vectorcontaining the gene of interest, before being injected in to therecipient.

[0148] Specifically, bone marrow stem cells were harvested from maledonor (4-8 weeks old) BALB/c AnNCr mice (National Cancer Institute,Division of Cancer Treatment Animal Program, Frederick, Md.). The cellswere plated at a density of 1-2×10⁷ cells/10 cm dish and cultured for 48hours in Dulbecco's modified Eagle's medium (DMEM) containing; 10%heat-inactivated fetal bovine serum, glutamine, Pen/Strep, 100 U/ml ofinterleukin-6 (IL-6) and stem cell factor (SCF; Immunex, Seattle, Wash.)to stimulate cell growth (Schiffmann, et. al., 1995).

[0149] Concurrently, a viral package cell line was cultured for 24hours. The package cell line used by Schiffmann, et al. was GP+E86 andthe viral vector was the LG retroviral vector based on the LN series ofretroviral vectors.

[0150] After the appropriate incubation period, 1-2×10⁷ stem cells wereplated on a 10 cm dish containing the viral package cells andco-cultured for 48 hours in the presence of 8 μg/ml of polybrene andunder the same growth factor stimulation conditions as the donor stemcells. The stem cells were then harvested, washed of growth media andinjected into recipient mice at dosages of 2×10⁷ cells/injection formultiple injections (total of 5 injections either daily or weekly).

[0151] Successful stem cell transduction and engraftment of stem cellscan be determined through, for example, PCR analysis, immunocytochemicalstaining, Southern Northern or Western blotting, or by other suchtechniques known to one skilled in the art.

[0152] Mammals

[0153] Mammals that are useful according to the invention include anymammal (for example, human, mouse, rat, sheep, rabbit, goat, monkey,horse, hamster, pig or cow). A non-human mammal according to theinvention is any mammal that is not a human, including but not limitedto a mouse, rat, sheep, rabbit, goat, monkey, horse, hamster, pig or acow.

[0154] Dosage and Mode of Administration

[0155] By way of example, a patient in need of pancreatic stem cells asdescribed herein can be treated as follows. Cells of the invention canbe administered to the patient, preferably in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle, byingestion, injection, inhalation or any number of other methods. Apreferred method is endoscopic retrograde injection. Another preferredmethod is injection or placement of the cells or pseudo-islet likeaggregates into the space under the renal capsule. The dosagesadministered will vary from patient to patient; a “therapeuticallyeffective dose” can be determined, for example but not limited to, bythe level of enhancement of function (e.g., insulin production or plasmaglucose levels). Monitoring levels of stem cell introduction, the levelof expression of certain genes affected by such transfer, and/or thepresence or levels of the encoded product will also enable one skilledin the art to select and adjust the dosages administered. Generally, acomposition including stem cells will be administered in a single dosein the range of 10⁵-10⁸ cells per kg body weight, preferably in therange of 10 ⁶-10⁷ cells per kg body weight. This dosage may be repeateddaily, weekly, monthly, yearly, or as considered appropriate by thetreating physician. The invention provides that cell populations canalso be removed from the patient or otherwise provided, expanded exvivo, transduced with a plasmid containing a therapeutic gene ifdesired, and then reintroduced into the patient.

[0156] Pharmaceutical Compositions

[0157] The invention provides for compositions comprising a stem cellaccording to the invention admixed with a physiologically compatiblecarrier. As used herein, “physiologically compatible carrier” refers toa physiologically acceptable diluent such as water, phosphate bufferedsaline, or saline, and further may include an adjuvant. Adjuvants suchas incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,or alum are materials well known in the art.

[0158] The invention also provides for pharmaceutical compositions. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carrier preparationswhich can be used pharmaceutically.

[0159] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for ingestion by the patient.

[0160] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0161] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

[0162] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0163] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer' solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0164] For nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0165] The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

[0166] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc . . . Saltstend to be more soluble in aqueous or other protonic solvents that arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%sucrose, 2%-7% mannitol at a Ph range of 4.5 to 5.5 that is combinedwith buffer prior to use.

[0167] After pharmaceutical compositions comprising a compound of theinvention formulated in a acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition with information including amount, frequency andmethod of administration.

[0168] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples, which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLE 1

[0169] Isolation of Nestin-Positive Stem Cells from Rat Pancreas.

[0170] Rat islets were isolated from the pancreata of 2-3 month oldSprague-Dawley rats using the collagenase digestion method described byLacy and Kostianovsky. Human islets were provided by the DiabetesResearch Institute, Miami, Fla. using collagenase digestion. The isletswere cultured for 96 hrs at 37° C. in 12-well plates (Falcon 3043plates, Becton Dickinson, Lincoln Park, N.J.) that had been coated withconcanavalin A. The culture medium was RPMI 1640 supplemented with 10%fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, 100 ,g/mlstreptomycin, 100 units/ml penicillin, 0.25 μg/ml amphotericin B (GIBCOBRL, Life Science Technology, Gaithersburg, Md.), and 71.5 mMP-mercaptoethanol (Sigma, St. Louis, Mo.).

[0171] After 96 hrs, fibroblasts and other non-islet cells had adheredto the surface of concanavalin A coated wells and the islets remainedfloating (did not adhere to the surface). At this time, the mediacontaining the islets were removed, centrifuged down, and the purgedislets replated in 12-well plates without a coating of concanavalin A.The islets were then cultured in the above RPMI 1640 medium supplementedwith 20 ng/ml of basic fibroblast growth factor-2 and 20 ng/ml ofepidermal growth factor.

[0172] The islets adhered to the surface of the plates, and cells grewout and away from the islets in a monolayer. These cells that form amonolayer were nestin-positive by immunostaining with a rabbit anti-ratnestin antiserum developed by Dr. Mario Vallejo at the MassachusettsGeneral Hospital. Other nestin antibodies may be used, for example theR.401 antibody described hereinabove, or the MAB533 antibody. Amonoclonal antibody specific for rat embryo spinal cord nestin, MAB353,ATCC No. 1023889; is described in Journal of Neuroscience 1996;16:1901-100; and also available from Chemicon International, Single OakDr., Temecula, Calif. 92590 USA. After two weeks of culture, several(3-5) of the nestin-positive monolayer cells were removed by pickingwith a capillary tube (cylinder cloning) and were replated on the12-well plates (not coated with concanavalin A) and cultured in the RPMI1640 medium further supplemented with bFGF-2 and EGF. The cellspropagated at a rapid rate and reached confluence after six days ofculture. After 12 days of culture, the cell monolayer formed waves inwhich they begin to pile up in a co-linear manner. On day 15 of culture,the cell waves began to condense, migrate into spheroid bodies and byday 17 the surface of the wells contained these spheroid bodies (ca. 100μm in diameter), empty spaces, and a few areas of remaining monolayercells. Several of these monolayer cells were re-picked and re-cloned andthe process described above occurred again in precisely the sametemporal sequence.

EXAMPLE 2

[0173] Differentiation of Pancreatic Stem Cells to Form Islet

[0174] Pancreatic islets from rats were first cultured in RPMI mediumcontaining 10% fetal bovine serum using concanavalin-A coated 12-wellplates. The islets were maintained in culture for three days in theabsence of added growth factors other than those supplied by fetalbovine serum. After this period, during which the islets did not attach,the islets were transferred to fresh plates without concanavalin A. Thestem cells were then stimulated to proliferate out from the islets as amonolayer by exposing them to bFGF-2 (20 ng/ml) and EGF (20 ng/ml) for24 days. After the 24 day period, the monolayer was confluent. Amongthem was a population of cells surrounding the islets. Cells from thatpopulation were picked and subcloned into new 12-well plates and againcultured in the medium containing bFGF and EGF. The subcloned cellsproliferated rapidly into a monolayer in a clonal fashion, expandingfrom the center to the periphery. The cells became confluent at day 6and then started to form a wave of overlapping cells on day 12. By day17 the cells migrated almost entirely into spherical structures andtubular structures resembling islet-like structures (pseudo-islet likeaggregates) and duct-like structures (pseudo-ducts) (FIG. 4). RT-PCRanalysis revealed that the pseudo-islet like aggregates were expressingNCAM (a marker for endocrine cells, see FIG. 5), cytokeratin 19 (amarker for ductal cells, see FIG. 5), and the transcription factorbrain-4 (a beta cell marker). Treatment with growth factors is requiredto achieve terminal differentiation to mature islet cells.

EXAMPLE 3

[0175] Isolation and culture of human or rat pancreatic islets

[0176] Human pancreatic islets were isolated and cultured. Human islettissue was obtained from the islet distribution program of the CellTransplant Center, Diabetes Research Institute, University of MiamiSchool of Medicine and the Juvenile Diabetes Foundation Center for IsletTransplantation, Harvard Medical School, Boston, Mass. Thoroughly washedislets were handpicked, suspended in modified RPMI 1640 media (11.1 mMglucose) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 1mM sodium pyruvate, 100 U per mL penicillin G sodium, 100 μg per mLstreptomycin sulfate, 0.25 ng per mL amphotericin B, and 71.5 PMβ-mercaptoethanol, and added to Falcon 3043 12-well tissue cultureplates that had been coated with Concanavalin A (ConA). The isletpreparation was incubated for 96 hrs at 37° C. with 95% air and 5% CO₂.In these conditions, many islets remained in suspension (floated),whereas fibroblasts and other non-islet cells attached to thesubstratum. After 96 h of incubation the media containing the suspendedislets was carefully removed, the islets were manually picked andresuspended in the modified RPMI 1640 media now further supplementedwith 20 ng/mL each of basic fibroblast growth factor (bFGF) andepidermal growth factor (EGF). The islet suspension (containing 20-30islets per well) was added to 12-well tissue culture plates not coatedwith ConA. The islets immediately adhered to the surfaces of the plates.Within several days, a monolayer of cells was observed growing out andaway from the islets. In certain instances, human-derived cells werecultured in modified RPMI media containing 2.5 mM glucose, and severalgrowth factor combinations that include activin-A (2 nM), hepatocytegrowth factor (100 pM), or betacellulin (500 pM). In those experimentsin which cells were challenged with 10 mM nicotinamide, the mediacontained no serum or growth factors.

EXAMPLE 4

[0177] Effects of Glucose and GLP-1 on Differentiation of PancreaticStem Cells.

[0178] Elevation of plasma glucose concentration leads to increasedpancreatic islet size. The effect of the glucose concentration in theculture medium was therefore investigated using isolated islets, whichcontain nestin-positive stem cells. Rat pancreatic islets were culturedin a medium containing high (16.7 mM) glucose or in normal (5.6 mM)glucose. After four days, RT-PCR was performed to determine the level ofnestin mRNA. The results indicated a three-fold stimulation of nestinmRNA levels in the islets cultured in high glucose compared to theislets cultured in normal glucose (FIG. 6).

[0179] Similarly, injection of glucagon-like peptide-1 (GLP-1) into micewas found to increase islet mass by 2-fold in 48 hours. Knockout micehaving a disrupted gene for GLP-1 receptor were examined for nestinexpression in pancreatic islets. Immunostaining using a nexin antibodywas found to be markedly reduced compared to normal mice with GLP-1receptors.

[0180] Animal Model of Diabetes Mellitus

[0181] Treatments for diabetes mellitus type that result in relief ofits symptoms are tested in an animal which exhibits symptoms ofdiabetes. It is contemplated that the animal will serve as a model foragents and procedures useful in treating diabetes in humans. Potentialtreatments for diabetes can therefore be first examined in the animalmodel by administering the potential treatment to the animal andobserving the effects, and comparing the treated animals to untreatedcontrols.

[0182] The non-obese diabetic (NOD) mouse is an important model of typeI or insulin dependent diabetes mellitus and is a particularly relevantmodel for human diabetes (see Kikutano and Makino, 1992, Adv. Immunol.52:285 and references cited therein, herein incorporated by reference).The development of type I diabetes in NOD mice occurs spontaneously andsuddenly without any external stimuli. As NOD mice develop diabetes,they undergo a progressive destruction of β-cells which is caused by achronic autoimmune disease. The development of insulin-dependentdiabetes mellitus in NOD mice can be divided roughly into two phases:initiation of autoimmune insulitis (lymphocytic inflammation in thepancreatic islets) and promotion of islet destruction and overtdiabetes. Diabetic NOD mice begin life with euglycemia, or normal bloodglucose levels, but by about 15 to 16 weeks of age the NOD mice startbecoming hyperglycemic, indicating the destruction of the majority oftheir pancreatic β-cells and the corresponding inability of the pancreasto produce sufficient insulin. In addition to insulin deficiency andhyperglycemia, diabetic NOD mice experience severe glycosuria,polydypsia, and polyuria, accompanied by a rapid weight loss. Thus, boththe cause and the progression of the disease are similar to humanpatients afflicted with insulin dependent diabetes mellitus. Spontaneousremission is rarely observed in NOD mice, and these diabetic animals diewithin 1 to 2 months after the onset of diabetes unless they receiveinsulin therapy.

[0183] The NOD mouse is used as an animal model to test theeffectiveness of the various methods of treatment of diabetes byadministering a stem cell preparation according to the invention. Assuch, treatment via administration of stem cells are tested in the NODmouse for their effect on type I diabetes.

[0184] The stem cells are administered to a NOD mouse, typicallyintraperitoneally, according to the following dosage amounts. NOD miceare administered about 1×10¹ to 1×10⁴ cells per mouse. Administration ofthe cells is started in the NOD mice at about 4 weeks of age, and iscontinued for 8 to 10 weeks, e.g., 3 times a week. The mice aremonitored for diabetes beginning at about 13 weeks of age, being testedtwice per week according to the methods described below. The effects oftreatment are determined by comparison of treated and untreated NODmice.

[0185] The effectiveness of the treatment methods of the invention ondiabetes in the NOD mice is monitored by assaying for diabetes in theNOD mice by means known to those of skill in the art, for example,examining the NOD mice for polydipsia, polyuria, glycosuria,hyperglycemia, and insulin deficiency, or weight loss. For instance, thelevel of urine glucose (glycosuria) can be monitored with Testape (EliLilly, Indianapolis, Ind.) and plasma glucose levels can be monitoredwith a Glucometer 3 Blood Glucose Meter (Miles, Inc., Elkhart, Ind.) asdescribed by Burkly, 1999, U.S. Pat. No. 5,888,507, herein incorporatedby reference. Monitoring urine glucose and plasma glucose levels bythese methods, NOD mice are considered diabetic after two consecutiveurine positive tests gave Testape values of +1 or higher or plasmaglucose levels >250 mg/dL (Burkly, 1999, supra). Another means ofassaying diabetes in NOD mice is to examine pancreatic insulin levels inNOD mice. For example, pancreatic insulin levels can be examined byimmunoassay and compared among treated and control mice (Yoon, U.S. Pat.No. 5,470,873, herein incorporated by reference). In this case, insulinis extracted from mouse pancreas and its concentration is determined byits immunoreactivity, such as by radioimmunoassay techniques, usingmouse insulin as a standard.

[0186] In addition to monitoring NOD mice for diabetes in general, theeffects of the inventive methods of treatment are also monitored forgene-specific or gene product-specific effects if the stem cellsadministered were transformed or transfected with a heterologous gene,thereby allowing a correlation to be drawn between expression of theheterologous gene and its effects on diabetes. For example, the presenceof the heterologous gene product may be examined by immunohistochemistryof the pancreatic β-cells of NOD mice for the gene product and forinsulin. The expression of the patched and smoothened genes is furtherexamined in NOD mouse islets by detection of the RNA transcript for thepatched and smoothened receptors. Reverse transcription-polymerase chainreaction (RT-PCR) amplification is performed by known means to amplify afragment of mouse patched or smoothened cDNA, and analyzed by agarosegel electrophoresis, according to standard means. The identification ofthe amplified cDNA fragment is confirmed as corresponding to the patchedor smoothened RNA by hybridization of the amplified fragment with aradiolabeled internal oligonucleotide probe for the patched orsmoothened genes, or by other such methods as known to one skilled inthe art.

EXAMPLE 5

[0187] Immunocytochemical Identification of Nestin Positive Human andRat Pancreatic Stem Cells

[0188] Pancreatic islets were analyzed for nestin expression. Islets andstem cells were isolated as described above. Nestin expression wasobserved by immunocytochemical staining in a distinct population ofcells within developing islet clusters of embryonic day 16 (E16) ratpancreas (FIG. 8A) and in islets of the adult pancreas (postnatal 60days) (FIG. 8B). Immunocytochemical staining was performed as follows.

[0189] Cryosections (6 μM) prepared from embryonic day 16 and adult (60day) rat pancreata as well as cells were fixed with 4% paraformaldehydein phosphate. Cells were first blocked with 3% normal donkey serum for30 min at room temperature and incubated with primary antisera overnightat 4° C. The antisera were rinsed off with PBS and incubated with therespective Cy-3 and Cy-2 labeled secondary antisera for 1 hour at roomtemperature. Slides were then washed with PBS and coverslipped withfluorescent mounting medium (Kirkegaard and Perry Labs, Gaithersburg,Md.). Tissue sections were incubated overnight at 4° C. with primaryantisera. Primary antisera were then rinsed off with PBS, and slideswere blocked with 3% normal donkey serum for 10 min at room temperaturebefore incubation with donkey anti-Cy3 (indocarbocyanine) and eitheranti-guinea pig (insulin), anti-mouse (glucagon), or anti-sheep(somatostatin) sera DTAF (Jackson Immuno Research Laboratories, WestGrove, Pa.) for 30 min at room temperature. Slides were then rinsed withPBS and coverslipped with fluorescent mounting medium (Kirkegaard andPerry Laboratories, Gaithersburg, Md.). Fluorescence images wereobtained using a Zeiss Epifluorescence microscope equipped with anOptronics TEC-470 CCD camera (Optronics Engineering, Goleta, Calif.)interfaced with a PowerMac 7100 installed with IP Lab Spectrum analysissoftware (Signal Analytics Corp, Vienna, Va.).

[0190] The nestin-positive cells are distinct from β-, α-, δ-, andPP-cells because they do not co-stain with antisera to the hormonesinsulin (FIG. 8A & B), glucagon, somatostatin, or pancreaticpolypeptide. The nestin-positive cells also do not co-stain withantisera to collagen IV, a marker for vascular endothelial cells (FIG.8C) nor with an antiserum to galanin, a marker for nerve cells or amonoclonal antibody to cytokeratin 19, a specific marker for ductalcells (FIG. 8). Nestin-positive staining is associated with distinctcells within the islets clearly observed by nuclear costaining (FIG.4D).

EXAMPLE 6

[0191] Identification of Nestin Positive Human and Rat Stem Cells byRT-PCR

[0192] To confirm the immunocytochemical identification of nestinexpression in pancreatic islets, we performed an RT-PCR of the nestinmRNA using total RNA prepared from freshly isolated rat islets and humanislet tissue. RT-PCR was performed according to the following method.

[0193] Total cellular RNA prepared from rat or human islets was reversetranscribed and amplified by PCR for 35 cycles as described previously(Daniel et al., 1998, Endocrinology, 139:3721-3729). Theoligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are: Rat nestin: Forward,5′gcggggcggtgcgtgactac3′; Reverse, 5′aggcaagggggaagagaaggatgt3′;Hybridization, 5′aagctgaagccgaatttccttgggataccagagga3′. Rat Forward,5′acagccagtacttcaagacc3′; keratin 19: Reverse, 5′ctgtgtcagcacgcacgtta3′;Hybridization, 5′tggattccacaccaggcattgaccatgcca3′. Rat NCAM: Forward,5′cagcgttggagagtccaaat3′; Reverse, 5′ttaaactcctgtggggttgg3′;Hybridization, 5′aaaccagcagcggatctcagtggtgtggaacgatgat3′. Rat IDX-1Forward, 5′atcactggagcagggaagt3′ Reverse, 5′gctactacgtttcttatct3′Hybridization, 5′gcgtggaaaagccagtggg3′ Human Forward,5′agaggggaattcctggag3′; nestin: Reverse, 5′ctgaggaccaggactctcta3′;Hybridization, 5′tatgaacgggctggagcagtctgaggaaagt3′. Human Forward,5′cttttcgcgcgcccagcatt3′; keratin: Reverse, 5′gatcttcctgtccctcgagc3′;Hybridization, 5′aaccatgaggaggaaatcagtacgctgagg3′. Human Forward,5′atctggactccaggcgtgcc3′; glucagon: Reverse, 5′agcaatgaattccttggcag3′;Hybridization, 5′cacgatgaatttgagagacatgctgaaggg3′.

[0194] Primers were selected from two different exons and encompassed atleast one intronic sequence. In addition, an RT minus control was runfor most samples. PCR cycling was at 94° C. for 1 min followed by 94° C.for 10 secs, 58/56° C. for 10 secs, 72° C. for 1 min, 35 cycles, and 72°C. for 2 min. The annealing temperature was 58° C. for rat nestin and56° C. for the remaining primer pairs.

[0195] For Southern hybridization oligonucleotide probes wereradiolabeled with T4 polynucleotide kinase and γ³²P ATP. Radiolabeledprobes were hybridized to PCR products that had been transferred tonylon membranes at 37° C. for one hour, then washed in 1×SSC+0.5% SDS at55° C. for 10-20 min or 0.5×SCC+0.5% SDS at 42° for the human PCRproducts.

[0196] The RT-PCR generated products of the correctly predicted size(FIG. 8E, upper panels) and were confirmed by Southern blotting (FIG.8E, lower panels) and by DNA sequencing of the products. These datademonstrate the identification of a new cell type in pancreatic isletsthat expresses nestin and may represent an islet pluripotential stemcell similar to the nestin-positive stem cells in the central nervoussystem.

EXAMPLE 7

[0197] In Vitro Proliferation of Nestin Positive Stem Cells

[0198] The ability of nestin-positive stem cells to proliferate in vitrowas determined.

[0199] Islets prepared from 60 day-old rats or a normal adult human werefirst plated on concanavalin-A-coated dishes and cultured in modifiedRPMI 1640 medium containing 10% fetal bovine serum for four days topurge the islet preparation of fibroblasts and other non-islet cellsthat adhered to the ConA-coated plates. The islets that did not adhereto the plates under these culture conditions were collected andtransferred to 12-well plates (without ConA coating) containing the samemodified RPMI 1640 medium now additionally supplemented with bFGF andEGF (20 ng/mL each). The growth factors bFGF and EGF together wereselected because they are known to stimulate the proliferation of neuralstem cells derived from ependyma of the brain (Reynolds and Weiss, 1996,Dev. Biol., 175:1-13). The islets attached to the plates and cellsslowly grew out of the islet as a monolayer (estimated cell doublingtime 40-45 hrs in human cells). The outgrowing monolayer of cells werephenotypically homogenous (FIG. 9A, panel 1) and expressed nestin (FIG.9A, panel 2). Rat cells were picked from the monolayer (batches of atleast 20-30 cells), subcloned into 12-well plates, and incubated withthe modified RPMI 1640 medium (11.1 mM glucose) containing bFGF and EGF.The subcloned cells grew rapidly and became confluent at six days withan estimated cell doubling time of 12-15 hrs (FIG. 9A, panel 3), and by12 days formed wave-like structures. After 15-17 days of culture, thecells formed islet-like clusters (ILCs) (FIG. 9A, panel 4). Similarcells were cloned from human islets (FIG. 9B). Upon reaching confluence(FIG. 9B, panel 1), the human cells migrated to form large vacuolatedstructures in the dish (FIG. 9B, panels 2 and 3). The cells lining thelarge spaces then changed morphology, rounded, and aggregated togetherforming three dimensional ILCs (FIG. 9B, panels 4-6).

[0200] Indicators of differentiation of these nestin-positive isletprogenitor cells (NIPs) that formed these ILCs were characterized byRT-PCR and Southern blot and found that they express the endocrinemarker NCAM (neural cell adhesion molecule) (Cirulli et al., 1994, J.Cell Sci., 107:1429-36) (FIG. 9C, right panel) and the ductal cellmarker CK19 (cytokeratin 19) (Bouwens et al., 1998, J. Pathol.,184:234-9; Bouwens et al., 1995, J. Histochem. Cytochem., 43:245-53;Bouwens et al., 1994, Diabetes, 43:1279-93) (FIG. 9C, left panels). Atthis stage of the studies it was concluded that when the NIPs becameconfluent and aggregated into islet-like cell clusters, they began toexpress pancreatic genes (NCAM and CK19), but were limited in expressionof islet genes because of the absence of growth factors essential fortheir differentiation to endocrine cells. It was also recognized thatthe differentiation of a progenitor cell population typically requiresfirst a proliferative phase and then quiescence of proliferation in thepresence of differentiation-specific morphogen growth factors. Thereforethe culture conditions were modified in some instances by replacing themedia containing 11.1 mM glucose, bFGF and EGF, which inducesproliferation of cells, with media containing lower glucose (2.5 mM),which is less proliferative, and the factors HGF/Scatter Factor orbetacellulin and Activin A. Glucose is a known proliferative factor forpancreatic islet β-cells (Swenne, 1992, Diabetologia, 35:193-201;Bonner-Weir, 1989, Diabetes, 38:49-53) and both HGF/Scatter Factor andActivin A have been shown to differ entiate the pancreatic ductal cellline AR42J into an endocrine phenotype that produces insulin, glucagon,and other pancreatic endocrine cell proteins (Mashima et al., 1996,Endocrinology, 137:3969-76; Mashima et al., 1996, J. Clin. Invest.,97:1647-54).

[0201] Cultures containing ILCs expressed the pancreas-specifichomeodomain protein IDX-1 by inununocytochemistry (FIG. 10A, upperpanel), RT-PCR and Southern blot (FIG. 10B), and by Western immunoblot(FIG. 10C). The ILCs also expressed the mRNA encoding proglucagon asseen by RT-PCR (FIG. 10D) and produced immunoreactive glucagon,glucagon-like peptide-1, and insulin. Radioimmunoassays of mediaobtained following 72-96 h of culture of islet-like clusters in severalwells gave values of 40-80 pg/ml GLP-1, 30-70 pg/ml glucagon, 29-44pg/ml insulin. Radioimmunoassays were performed as follows.

[0202] Insulin and glucagon concentrations in culture media weredetermined by ultra sensitive radioimmunoassay kits purchased from LincoResearch Inc. and DPC Inc., respectively. The antisera supplied in therespective kits are guinea pig anti-human insulin and rabbit anti-humanglucagon. GLP-1 secretion was measured with an anti-humanGLP-1(7-36)amide rabbit polyclonal antiserum raised by immunization of arabbit with a synthetic peptide CFIAWLVKGR amide conjugated to keyholelimpet hemocyanin. The antiserum is highly specific for the detection ofGLP-1(7-36)amide and only weakly detects proglucagon. The sensitivitylevels for these assays are 6 pg/mL, 13 pg/mL and 10.2 pg/mL,respectively.

[0203] Incubation of the ILCs for 7 days in 10 mM nicotinamide, asdescribed by Ramiya et al. (Ramiya et al., 2000, Nat. Med., 6:278-282),increased insulin secretion by 2-to 3-fold.

[0204] Several additional pancreatic markers were expressed indifferentiated NIPs such as glucose transporter-2 (Wang et al., 1998),synaptophysin, and HGF (Menke et al., 1999) as shown in FIG. 15. Todetermine whether the differentiating NIPs may have properties ofpancreatic exocrine tissue, we used RT-PCR and detected the expressionof amylase and procarboxypeptidase (FIG. 15).

[0205] Some cultures of NIPs containing stem cells also expressed themRNA encoding proglucagon and insulin as seen by RT-PCR (FIG. 16A andB).

[0206] The expression of IDX-1 is of particular importance because it isrecognized to be a master regulator of pancreas development, andparticularly to be required for the maturation and functions of thepancreatic islet β-cells that produce insulin (Stoffers et al., 1997,Trends Endocrinol. Metab., 8:145-151).

[0207] Because the neogenesis of new islets is also known to occur bydifferentiation of cells in pancreatic ducts, particularly during theneonatal period (rats and mice) but to some extent throughout adult life(Bonner-weir et al., 1993, Diabetes, 42:1715-1720; Rosenberg, 1995, CellTransplant, 4:371-383; Bouwens et al., 1996, Virchows Arch.,427:553-560), nestin expression was analyzed in the pancreatic ducts ofadult rats. By dual fluorescence immunocytochemistry with antisera tonestin and to cytokeratin 19, a marker of ductal epithelium, nestin isstrongly expressed in localized regions of both the large and smallducts, as well as in some centrolobular ducts within the exocrine acinartissue (FIGS. 11A and 11B). Remarkably, the localized regions of nestinexpression in the ducts are mostly devoid of staining with the anti CK19antiserum. Further, the nestin-positive cells in the ducts appear tohave a morphology that is distinct from that of the epithelial cells.The epithelial cells consist of a homogenous population of cuboidal,rounded cells, whereas the nestin-positive cells are nucleated,serpiginous and appear to reside in the interstices among or aroundepithelial cells (FIG. 11C).

[0208] Thus, CK19 is not expressed in the majority of ductal cells thatexpress nestin suggesting that these nestin-expressing cells locatedwithin the pancreatic ducts are a passenger population of cells distinctfrom the ductal epithelial cells and are stem cells that have not yetdifferentiated into a ductal or endocrine phenotype. The finding oflocalized populations of nestin-expressing cells within the pancreaticducts and islets of the adult rat pancreas further supports the ideathat rat pancreatic ducts contain cells that are progenitors of isletcells (neogenesis), but these progenitors are not a subpopulation ofductal epithelial cells per se.

EXAMPLE 8

[0209] Transplantation of Pancreatic Stem Cells Engineered to ExpressIDX-1 in Human Subjects with Diabetes Mellitus

[0210] Islets isolated from pig or human donor pancreata, or frompancreatic biopsy of eventual human transplant recipient are cultured exvivo in conditions that stimulate outgrowth of stem cells. Stem cellsare then isolated away from islets (cloned), expanded in vitro inproliferation media containing bFGF-2, EGF, and 11.1 mM glucose,transfected/injected with an expression vector containing DNA encodingtranscription factor IDX-1, and transplanted into a diabetic recipient.Alternatively, IDX-1-transfected stem cells are treated with GLP-1, orother differentiation morphogens or growth factors for 1-3 days beforetransplantation to initiate processes of differentiation of engineeredstem cells to β-cells. In one embodiment, stem cells are neitherexpanded or differentiated prior to administration to the recipient orare only expanded or differentiated prior to administration to therecipient. In one embodiment, GLP-1 is administered to the recipientduring and for several days after transplantation to stimulatedifferentiation of stem cells and encourage successful engraftment.According to this method, xenographs (pig islets) or allographs (humanislets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are carried out. It ishypothesized that when transplanted to a host recipient the stem cellgenetic repertoire is reprogrammed so that the host recognizes the stemcells (in the case of xenographs or allographs) as self, such thatimmune intolerance and graft rejection and destruction by autoimmunity(type 1 diabetes) does not occur.

EXAMPLE 9

[0211] Transplantation of Pancreatic Stem Cells Cultured to StimulateExpression of IDX-1 in Human Subjects with Diabetes Mellitus

[0212] Islets isolated as described are cultured ex vivo for severaldays in conditions that stimulate first the expansion (proliferation) ofstem cells that exist within the islets and then the expression oftranscription factor IDX-1. The proliferation of stem cells is achievedby culturing the islets in media containing bFGF-2, EGF, and 11.1 mMglucose. Induction of the expression of IDX-1 is achieved by incubationin the presence of GLP-1 and 2 mM glucose. The islets so preconditionedby the treatments described are transplanted to the host recipient.Additionally, the host recipient may be administered GLP-1 during andfor several days after the transplantation to further expand anddifferentiate stem cells to insulin-producing cells to enhance successof engraftment.

[0213] According to this method, xenographs (pig islets), allographs(human islets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are effectuated.

EXAMPLE 10

[0214] Xenogeneic Transplantation of Pancreatic Stem Cells into theKidney

[0215] Human nestin-positive-islet progenitor cells (NIPS) were isolatedas described, and transplanted under the renal capsules of eight C57B16mice that were not immunosuppressed. The transplanted human cells werenot rejected by the mouse recipient. Current understanding is that axenograft, such as human tissue, would be rejected by the mouse within5-10 days. Contrary to current understanding, we found that in 8 of the8 non-immunosuppressed mice tested to date, all of the transplantssuccessfully engrafted and proliferated into large masses of tissueengulfing the pole of the kidney by one month (30-38 days) after atransplantation of approximately 10⁵ to 10⁶ cells.

[0216] One C57B 16 mouse was sacrificed and determined to have a largearea of new growth at the site of transplantation. A section of thekidney that included the new tissue was divided into two pieces; onepiece was frozen for frozen section histology, and the other piece wasfixed in paraformaldehyde for paraffin section histology. Frozensections were prepared and stained with hematoxylin and eosin (H&E) andantisera to various islet cell antigens.

[0217] Examination of the H&E stained kidney section demonstrated thepresence of a new growth that was not part of the kidney, exhibiting apleiomorphic morphology consisting of a mixed mesenchymal and epithelialtissue containing hepatic, neural, ductal, adipodipic and hematopoeticcomponents. Photomicrographs of the kidney section demonstrated that thenew growth seemed to be invading the renal parenchyme, and theglomeruli. Specific immunostaining with human-specific (not mouse)antisera revealed cords of immunopositive cells staining forhuman-specific keratins, vimentin, and the CD45 leukocyte antigenspecific for human hematapoetic lymphocytes. The kidney of a secondC57B16 mouse also had a similar looking new growth at the site of theNIP transplantation.

[0218] The paraffin section of the NIP-engrafted kidney of a C57B16mouse (the first mouse to be sacrificed) was examined. The tissue blockthat was examined was from the top of the kidney and showed the foreigntissue to be well contained under the renal capsule with no signs of“invasion” into the renal parenchyma. Notably, amongst thepleiomorphic-looking graft tissues were areas that resembled renalparenchyma. Without being bound to theory one hypothesis is that thegraft consists of stem cells trying to differentiate and that the stemcells are not “invading” but simply migrating and proliferating andlooking for a niche, i.e. mesenchymal instructions. They may bereceiving cues from the kidney and may be attempting to differentiateinto kidney. The graft cells may not be malignant, but may be just stemcells attempting to carry out their function.

EXAMPLE 11

[0219] Xenogeneic Transplantation of Pancreatic Stem Cells into thePancreas

[0220] Human nestin-positive-islet progenitor cells (NIPS) are isolatedas described, and transplanted into the pancreas of mice that are notimmunosuppressed and are (a) injured by streptozotocin (to producestreptozotocin induced diabetes) treatment or (b) NOD mice in whichthere is an ongoing islet is with inflammation.

[0221] The pancreas of the transplanted animals is examined to determineif the NIPs find their proper niche, receive instructions from the isletregion, and differentiate into islet (P-cell) cells.

EXAMPLE 12

[0222] Treatment of Diabetes by Xenogeneic Transplantation of PancreaticStem Cells

[0223] Human islets are isolated as described and cultured for severaldays in vitro to expand the stem cell population. Human NIPS aretransplanted to the liver via the portal vein (according to conventionalprocedures well known in the art for transplantation to the liver.

[0224] Alternatively, a population of human NIPs (isolated as described)are introduced into the blood stream. In certain embodiments, the humanNIPS are introduced via the pancreatic artery, to direct them to thediabetic pancreas.

[0225] A population of control (untransplanted animals) and transplantedanimals are analyzed for amelioration of the symptoms of diabetes (e.g.blood glucose levels, insulin levels, number of pancreatic P-cells.

EXAMPLE 13

[0226] Identification of Nestin Positive Stem Cells in the Liver

[0227] Rat livers were isolated and frozen section were preparedaccording to methods known in the art and described herein.

[0228] Frozen sections of rat liver (6pM) were immunostained with arabbit polyclonal anti-nestin serum. The immunofluorescent signal wasdeveloped by reaction of anti-donkey IgG serum tagged with thefluorophore, Cy3 (yellow-orange color. Nestin-positive cells surroundinga possible large biliary duct are depicted in FIG. 13A. Clusters ofnestin positive cells surrounding several small biliary ducts aredepicted in FIG. 13B.

EXAMPLE 14

[0229] Differentiation of NIPs Toward Hepatic Phenotype

[0230] Because of the reported apparent commonalties between hepaticstem cells (oval cells), hepatic stellate cells, and progenitor cells inthe pancreas, and the observations that following some injuries, theregenerating pancreas undergoes liver metaplasia (Slack, 1995; Reddy etal., 1991; Bisgaard et al., 1991; Rao et al., 1996), we performed RT-PCRto detect liver-expressed genes in the stem cells. PCR products wereobtained for XBP-1, a transcription factor required for hepatocytedevelopment (Reimold et al., 2000), and transthyretin, a liver acutephase protein. Several other liver markers were also expressed such as(X-fetoprotein (Dabeva et al., 2000), E-Cadherin (Stamatoglou et al.,1992), c-MET (Ikeda et al., 1998), HGF (Skrtic et al., 1999) andsynaptophysin (Wang et al., 1998); see FIG. 15)) The expression ofproteins shared by the pancreas and liver, such as HGF andsynaptophysin, may reflect their common origin from the embryonicforegut endoderm, and represent differentiation toward either pancreaticor hepatic phenotypes.

[0231] References

[0232] Bisgaard, H. C. and Thorgeirsson, S. S. 1991. Evidence for acommon cell of origin for primitive epithelial cells isolated from ratliver and pancreas. J. Cell Physiol. 147:333-343.

[0233] Bjornson, C. R. et al. 1999. Turning brain into blood: ahematopoietic fate adopted by adult neural stem cells in vivo. Science283:534-537.

[0234] Boggs, S. S. 1990. Targeted gene modification for gene therapy ofstem cells. Int J. Cell Cloning 8:80-96.

[0235] Bouwens, L. et al. 1994. Cytokeratins as markers of ductal celldifferentiation and islet neogenesis in the neonatal rat pancreas.Diabetes 43:1279-1283.

[0236] Bouwens, L. 1998. Transdifferentiation versus stem cellhypothesis for the regeneration of islet beta cells in the pancreas.Microsc. Res. Tech. 43:332-336.

[0237] Cornelius, J. G., et al. 1997. In vitro-generation of islets inlong-term cultures of pluripotent stem cells from adult mouse pancreas.Horm. Metab. Res. 29:271-277.

[0238] Dahlstrand, J., et al. 1992. Characterization of the human nestingene reveals a close evolutionary relationship to neurofilaments. J.Cell Sci. 103:589-597.

[0239] Dabeva, M. D. et al., 2000. Proliferation and differentiation offetal liver epithelial progenitor cells after transplantation into adultrat liver. Am. J. Pathol. 156:2017-2031.

[0240] Hockfield, S., and McKay, R. D. 1985. Identification of majorcell classes in the developing mammalian nervous system. J. Neurosci.5:3310-3328.

[0241] Ikeda et al., 1998. Activated rat stellate cells express c-metand respond to hepatocyte growth factor to enhance transforming growthfactor beta 1 expression and DNA synthesis. Biochem Biophys Res Commun250:769-775.

[0242] Johansson, C. B. et al. 1999. Identification of a neural stemcell in the adult mammalian central nervous system. Cell 96:25-34.

[0243] Karlsson, S. 1991. Treatment of genetic defects in hematopoieticcell function by gene transfer. Blood 78(10): 2481-2492.

[0244] Lendahl, U., et al. 1990. CNS stem cells express a new class ofintermediate filament protein. Cell 60:585-595.

[0245] Miller, A. D. 1990. Retrovirus packaging cells. Hum Gene Therapy1:5.

[0246] Morshead, C. M. et al. 1994. Neural stem cells in the adultmammalian forebrain: a relatively quiescent subpopulation ofsubependymal cells. Neuron 13:1071-1082.

[0247] Rao. M. S. et al., 1996. Expression of transcription factors andstem cell factor precedes hepatocyte differentiation in rat pancreas.Gene Expr 6:15-22.

[0248] Reddy, J. K. et al., 1991. Pancreatic Hepatocytes. An in vivomodel for cell lineage in pancreas of adult rat. Dig. Dis. Sci.36:502-509.

[0249] Reimold, A. M. et al., 2000 An essential role in liverdevelopment for transcription factor XBP-1. Genes Dev. 14:152-7.

[0250] Reynolds, B. A. and Weiss, S. 1996. Clonal and populationanalyses demonstrate that an EGF-responsive mammalian embryonic CNSprecursor is a stem cell. Dev. Biol. 175:1-13.

[0251] Schiffmann, et. al. 1995. Transfer of the humanglucocerebrosidase gene into hematopoietic stem cells of nonablatedrecipients: successful engraftment and long-term expression of thetransgene. Blood 86(3): 1218-1227.

[0252] Skrtic, S., et al., 1999. Hepatocyte-stimulated expression ofhepatocyte growth formation (HGF) in cultured rat hepatic stellatecells. J. Hepatol. 30:115-124.

[0253] Slack, J. M. W., 1995, Developmental Biology of the pancreas.Development, 121:1569-1580.

[0254] Stamatoglou, S. C. et al., 1992. Temporal changes in theexpression and distribution of adhesion molecules during liverdevelopment and regeneration. J. Cell. Biol. 116:1507-1515.

[0255] Wang, Z. et al., 1998. GLUT2 in pancreatic islets: crucial targetmolecule in diabetes induced with multiple low doses of streptozotocinin mice. Diabetes 47:50-56.

[0256] Williams, D. A. 1990. Expression of introduced genetic sequencesin hematopoietic cells following retroviral-mediated gene transfer. Hum.Gene Therapy 1:229.

[0257] OTHER EMBODIMENTS

[0258] Other Embodiments are within the claims that follow.

We claim:
 1. A method of treating a patient with diabetes mellitus,comprising the steps of: (a) isolating a nestin-positive pancreatic stemcell from a pancreatic islet of a donor; and (b) transferring the stemcell into the patient, wherein the stem cell differentiates into aninsulin-producing cell.
 2. The method of claim 1 , wherein the patientserves as the donor for said stem cells of step a.
 3. The method ofclaim 1 wherein, prior to the step of transferring, the stem cell istreated ex vivo with an agent selected from the group consisting of EGF,bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-l, a nucleicacid molecule encoding IDX-1, betacellulin, activin A, TGF-β, andcombinations thereof.
 4. The method of claim 1 , wherein the step oftransferring is performed via endoscopic retrograde injection.
 5. Themethod of claim 1 additionally comprising the step of: (c) treating thepatient with an immunosuppressive agent.
 6. The method of claim 5 ,wherein the immunosuppressive agent is selected from the groupconsisting of FK-506, cyclosporin, and GAD65 antibodies.
 7. A method oftreating a patient with diabetes mellitus, comprising the steps of: (a)isolating a nestin-positive pancreatic stem cell from a pancreatic isletof a donor; (b) expanding the stem cell ex vivo to produce a progenitorcell; and (c) transferring the progenitor cell into the patient, whereinthe progenitor cell differentiates into an insulin-producing beta cell.8. The method of claim 7 , wherein the patient serves as the donor forsaid stem cells of step a.
 9. The method of claim 7 , wherein the stepof expanding is performed in the presence of an agent selected from thegroup consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1,exendin-4, IDX-1, a nucleic acid molecule encoding IDX-l, betacellulin,activin A, TGF-β, and combinations thereof.
 10. The method of claim 7 ,wherein the step of transferring is performed via endoscopic retrogradeinjection.
 11. The method of claim 7 additionally comprising the stepof: (d) treating the patient with an immunosuppressive agent.
 12. Themethod of claim 11 , wherein the immunosuppressive agent is selectedfrom the group consisting of FK-506, cyclosporin, and GAD65 antibodies.13. A method of treating a patient with diabetes mellitus, comprisingthe steps of: (a) isolating a nestin-positive pancreatic stem cell froma pancreatic islet of a donor; (b) expanding the stem cell to produce aprogenitor cell; (c) differentiating the progenitor cell in culture toform pseudo-islet like aggregates; and (d) transferring the pseudo-isletlike aggregates into the patient.
 14. The method of claim 13 , whereinthe patient serves as the donor for said stem cells of step a.
 15. Themethod of claim 13 , wherein the step of expanding is performed in thepresence of an agent selected from the group consisting of EGF, bFGF-2,high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acidmolecule encoding IDX-l, betacellulin, activin A, TGF-β, andcombinations thereof.
 16. The method of claim 13 , wherein the step oftransferring is performed via endoscopic retrograde injection.
 17. Themethod of claim 13 additionally comprising the step of: (e) treating thepatient with an immunosuppressive agent.
 18. The method of claim 17 ,wherein the immunosuppressive agent is selected from the groupconsisting of FK-506, cyclosporin, and GAD65 antibodies.
 19. A method ofisolating a stem cell from a pancreatic islet of Langerhans, comprisingthe steps of: (a) removing a pancreatic islet from a donor; (b)culturing cells from the pancreatic islet; and (c) selecting anestin-positive clone from the culture.
 20. The method of claim 19 ,wherein the culturing is first performed in a vessel coated withconcanavalin A and then again performed in a vessel not coated withconcanavalin A.
 21. The method of claim 19 comprising the additionalstep of: (d) expanding the nestin-positive clone by treatment with anagent selected from the group consisting of EGF, bFGF-2, high glucose,KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encodingIDX-1, betacellulin, activin A, TGF-β, and combinations thereof.
 22. Amethod of inducing the differentiation of a nestin-positive pancreaticstem cell into a pancreatic progenitor cell, comprising the step of:treating a nestin-positive pancreatic stem cell with an agent selectedfrom the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF,IDX-1, a nucleic acid molecule encoding IDX-l, GLP-1, exendin-4,betacellulin, activin A, TGF-β, and combinations thereof, whereby thestem cell subsequently differentiates into a pancreatic progenitor cell.23. The method of claim 22 , wherein the pancreatic progenitor cellsubsequently forms pseudo-islet like aggregates.
 24. An isolated,nestin-positive human pancreatic or liver stem cell that is not a neuralstem cell.
 25. The isolated stem cell of claim 24 that differentiates toform insulin-producing beta cells.
 26. The isolated stem cell of claim24 that differentiates to form glucagon-producing alpha cells.
 27. Theisolated stem cell of claim 24 that differentiates to form pseudo-isletlike aggregates.
 28. The isolated stem cell of claim 24 thatdifferentiates to form hepatocytes.
 29. The isolated stem cell of claim24 that does not express class I MHC antigens.
 30. A method ofidentifying a pancreatic cell as a stem cell, comprising the step of:contacting a cell with a labeled nestin-specific antibody, whereby ifthe cell becomes labeled with the antibody the cell is identified as astem cell.
 31. The method of claim 30 further comprising the step of:contacting the cell with a labeled cytokeratin-19 specific antibody,whereby if the cell does not become labeled with the antibody the cellis identified as a stem cell.
 32. The method of claim 30 or 31 furthercomprising the step of: contacting the cell with a labeled collagen IVspecific antibody, whereby if the cell does not become labeled with theantibody the cell is identified as a stem cell.
 33. A method of inducinga nestin-positive pancreatic stem cell to differentiate intohepatocytes, comprising the step of: treating the nestin-positivepancreatic stem cell with an effective amount of an agent that inducesthe stem cell to differentiate into hepatocytes or into progenitor cellsthat differentiate into hepatocytes.
 34. The method of claim 33 ,wherein the agent is cyclopamine.
 35. A method of treating a patientwith liver disease, comprising the steps of: (a) isolating anestin-positive pancreatic stem cell from a pancreatic islet of a donor;and (b) transferring the stem cell into the patient, wherein the stemcell differentiates into a hepatocyte.
 36. The method of claim 35 ,wherein the patient serves as the donor for said stem cells of step a.37. A method of treating a patient with liver disease, comprising thesteps of: (a) isolating a nestin-positive pancreatic stem cell from apancreatic islet of a donor; (b) expanding the stem cell ex vivo toproduce a progenitor cell; and (c) transferring the progenitor cell intothe patient, wherein the progenitor cell differentiates into ahepatocyte.
 38. The method of claim 37 , wherein the patient serves asthe donor for said stem cells of step a.
 39. A method of treating apatient with liver disease, comprising the steps of: (a) isolating anestin-positive pancreatic stem cell from a pancreatic islet of a donor;(b) differentiating the stem cell ex vivo to produce a hepatocyte; and(c) transferring the hepatocyte into the patient.
 40. The method ofclaim 39 , wherein the patient serves as the donor for said stem cellsof step a.
 41. A pharmaceutical composition comprising the isolated stemcell of claim 24 admixed with a physiologically compatible carrier.